ES2775451T3 - Compositions of coated microvesicles derived from edible plants and methods for using them - Google Patents

Abstract

Composition, comprising: a microvesicle derived from an edible plant; and a plasma membrane that lines the microvesicle, the plasma membrane derived from a recognition cell.

Description

DESCRIPTION

Compositions of coated microvesicles derived from edible plants and procedures for using the same

REQUESTS RELATED TO GOVERNMENT INTEREST

The present invention was made with government support under grant number UH2TR000875 granted by the National Institutes of Health. The government has certain rights in the present invention.

TECHNICAL SECTOR

The subject matter disclosed herein relates to coated microvesicle compositions derived from edible plants and to methods for using the same for the diagnosis and treatment of disease. In particular, the subject matter disclosed herein relates to compositions that include edible plant-derived microvesicles that have a targeting cell-derived plasma membrane coating and that are useful in treating disease.

STATE OF THE PRIOR ART

Inflammation is a feature of most diseases, including cancer, autoimmune disease, and infectious disease. Development of specific delivery systems of target to inflammatory sites is urgently needed. The attraction of leukocytes, including T cells, to sites of inflammation and infection is an essential component of the host response to disease, including autoimmune and chronic inflammatory diseases, as well as infectious diseases and cancer. Recruitment of circulating T cells to sites of entry for pathogens or inflammation involves at least two separate migration processes, termed extravasation and chemotaxis. Adhesion to the luminal aspect of blood vessels, transendothelial migration, and subsequent chemotaxis of leukocytes are highly complex processes. Chemokines and their receptors play a coordinating role in the homeostatic circulation of T cells, as well as their movement to sites of inflammation or injury. Once T cells are within inflammatory tissue, their response can be affected by the many inflammatory chemokines that are overexpressed and have broad target cell selectivity. The fact that there is a redundancy within the chemokine network with respect to ligand-receptor binding and that a set of chemokines is overexpressed by a variety of cells in inflammatory tissues makes the use of chemokines a potential component for the development of therapeutic recognition agents.

For a therapeutic agent to exert its desired effect it needs to (1) reach the desired site and (2) be in physical contact with its target. The development of target-specific delivery systems has not yet been largely successful. Despite the many potential advantages to the use of nanoparticles and liposomes, obstacles to their use include cytotoxicity, induction of chronic inflammation, host immune responses, difficulties in large-scale production at affordable prices, and potential biological hazards to the environment. Unlike the situation with artificially synthesized nanoparticles, naturally released nano-sized exosomes derived from many different types of mammalian cells play an important role in intercellular communication. Nano-sized exosomes released from mammalian cells have been used for the encapsulation of drugs and siRNA to treat diseases in mouse models of disease without side effects. Although this approach is promising, producing large quantities of mammalian cell nanoparticles and assessing their potential biohazards has been challenging. Recently, exosome-like nanoparticles have been identified from edible plant tissue, including grapefruit, grapes and tomatoes, and have been produced in large quantities. See, for example, WO2013 / 070324. As with mammalian exosomes, nanoparticles similar to grape exosomes were shown to naturally encapsulate RNA, proteins and small lipids. Using in vitro cell culture models as well as mouse models, grapefruit NVGs were shown to be highly effective in delivering a variety of therapeutic agents, including drugs, DNA expression vectors, siRNA, and antibodies. . Despite the promise of these nanovectors, however, efficient and effective targeting of nanovectors to inflamed and / or diseased tissue remains a problem.

Patent WO2013 / 038734 discloses compositions comprising vesicles to deliver therapeutic molecules for the treatment of cardiovascular disease.

CHARACTERISTICS OF THE INVENTION

The subject matter disclosed herein meets some or all of the previously identified needs, as will be apparent to a person skilled in the art after studying the information disclosed herein.

These Features describe various embodiments of the subject matter disclosed herein and, in many cases, list variations and permutations of these embodiments. These Features merely include examples of the numerous and varied embodiments. The mention of one or more representative characteristics of a certain embodiment is also by way of example. Such an embodiment can usually exist with or without the mentioned feature (s); similarly, these features can be applied to other embodiments of the subject matter disclosed herein, whether or not it appears in these Features. To avoid excessive repetition, these Features do not list or suggest all possible combinations of those features.

The subject matter disclosed herein includes coated microvesicle compositions derived from edible plants and the use thereof for the treatment of an inflammatory disorder and / or cancer. In particular, the subject matter disclosed herein includes compositions that include a microvesicle derived from an edible plant; and a plasma membrane that lines the microvesicle, the plasma membrane derived from a recognition cell.

In the matter disclosed herein, a composition is disclosed comprising a microvesicle derived from an edible plant, wherein the microvesicle, in turn, is coated with a plasma membrane derived from a recognition cell. In some embodiments, the edible plant is a fruit or a vegetable, such as, in some embodiments, a grape, grapefruit, or tomato. In some embodiments, the target cell used to bypass the plasma membrane lining the microvesicle is an activated leukocyte.

Additionally, with respect to microvesicle compositions, in some embodiments, the microvesicle encapsulates a therapeutic agent. In some embodiments, the therapeutic agent is selected from a phytochemical agent and a chemotherapeutic agent. In some embodiments, the phytochemical agent is selected from curcumin, resveratrol, baicalein, equol, fisetin, and quercetin. In some embodiments, the chemotherapeutic agent is selected from the group consisting of retinoic acid, 5-fluorouracil, vincristine, actinomycin D, adriamycin, cisplatin, docetaxel, doxorubicin, and taxol. In other embodiments, the therapeutic agent comprises a nucleic acid molecule, such as, in some embodiments, a siRNA, a microRNA, or a mammalian expression vector. In some embodiments, pharmaceutical compositions are further disclosed in which the microvesicle compositions are combined with a pharmaceutically acceptable vehicle, carrier, or excipient.

Also disclosed in some embodiments of the subject matter disclosed herein is a composition, as described above, for use in treating an inflammatory disorder. In some embodiments, treating an inflammatory disorder comprises the step of administering to an individual in need thereof an effective amount of a microvesicle composition described herein. In some embodiments, the inflammatory disorder is selected from the group comprising sepsis, septic shock, colitis, colon cancer, and arthritis. The composition can be administered orally or intranasally. In some embodiments, after administration, the composition reduces the amount of an inflammatory cytokine in an individual, such as, in certain embodiments, tumor necrosis factor α, interleukin-1 p, interferon y, and interleukin-6.

In some embodiments of the matter disclosed herein, the composition, as described above, is for use in treating cancer in an individual. In some embodiments, treating a cancer in an individual comprises administering to an individual an effective amount of a microvesicle composition disclosed herein. In some embodiments of treating cancer, the therapeutic agent is selected from a phytochemical agent and a chemotherapeutic agent. In some embodiments, the cancer is selected from a brain cancer, a breast cancer, a lung cancer, and a colon cancer.

Other features and advantages of the present invention will be apparent to a person skilled in the art after studying the description, figures and non-limiting examples herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A-1D include schematic diagrams, graphs, and images showing characterization of NGV grapefruit-derived nanovectors coated on inflammatory cell plasma membrane (IGNV). Figure 1A is a schematic diagram showing the process of preparing the IGNVs and drug-loaded GNV microvesicles for the targeted delivery of therapeutic agents to inflammatory sites. Figure 1B is a graph showing the size distribution and surface Zeta potential of free GNV and EL4 cell plasma membrane coated GNV (IGNV) measured using a Zetasizer. Figure 1C includes images showing free GNV (left) and IGNV (right) that were visualized and imaged by scanning electron microscopy. Figure 1D includes images showing the colocalization of EL4 cell-derived plasma membranes and GNV nuclei, where, for the assembly of IGNV, EL4 cell-derived plasma membranes were labeled with PKH67 green dye and GNV nuclei were marked with PKH26 red dye, where 4T1 cells were cultured in the presence of GNV (upper panel) or IGNV (lower panel) for 12 hours, and where representative images of the cells were then taken using a confocal microscope with a magnification 400x. Figure 1E is a graph showing FRET-based measurements of IGNV formation, where DiO-labeled GNV and DPA-labeled membrane vesicles (n = 3) were mixed, where the mixture was subsequently extruded 20 times through a 200 nm polycarbonate porous membrane using an Avanti mini extruder or blending without additional extrusion was used as a control, and where extrudates and blended products were then diluted and fluorescence intensity measured.

Figures 2A-2E include images and graphs showing the ability of IGNV to utilize the membrane-dependent pathways of activated leukocytes and effectively target and deliver to inflammatory sites. Figure 2A includes images and graphs showing the results of a transwell assay for the detection of chemotaxis of plasma membrane-coated VNG from EL4 cells, where HUVEC cells were cultured in the upper chamber and 4TO7 cells were cultured in the lower chamber of a transwell plate, where transmigration of PKH26-labeled GNV or IGNV was imaged after 24 hours and 48 hours in culture using a confocal microscope, and where the intensity of the fluorescent signal of the media in the lower chamber was measured (n = 3) and was expressed as the percentage of efficiency in the transwell of the fluorescence intensity of GNV or IGNV labeled with PKH26. Figures 2B-2E are images and graphs showing the distribution of DiR dye labeled IGNV in: mouse model with LPS-induced acute inflammatory skin disease (Figure 2B); mice with DSS-induced colitis (Figure 2C); CT26 tumor model (Figure 2D); and 4T1 tumor model (Figure 2E); where live mouse images (left) were collected 6 hours and 24 hours after I.V. injection. of IGNVs labeled with DiR dye, where skin, colon and tumor tissues were removed 24 hours after injection and DiR dye signals were tracked, and where a representative image of each group of mice is shown (left panels ) and followed by graphical figures (right panels) that are presented as the average net intensity (intensity of sum / area, n = 5) (** p <0.01 and *** p <0.001).

Figures 3A-3I include images and graphs showing the ability of chemokine-mediated pathways to play a causal role in efficient targeted delivery of IGNV to inflammatory sites. Figure 3A includes images showing chemokine expression in normal skin (Normal), LPS-induced acute inflammatory skin tissues (Skin-LPS), tissues with CT26 (CT26 tumor) and 4T1 (4T1 tumor), where the expressions using a Proteome Profiler from R&D Systems, and where each point represents a chemokine detected by a capture antibody and printed in duplicate on a membrane. Figure 3B includes graphs showing chemokine receptor expression in IGNV, where IGNVs were analyzed by FACS analysis of IGNV coated on 4 pmi diameter aldehyde / sulfate latex beads. Figure 3C is a graph showing the in vitro transmigration of IGNV, where HUVEC cells (n = 3) were cultured in the upper chamber coated with fibronectin as a transmigration barrier, where IGNV labeled with PKH26 (PKH26-IGNV) were pre-incubated with Recombinant chemokines, as indicated in Figure 1C or with the 4T1 tumor extract, where, after washing, the pre-incubated PKH26-IGNVs were added to the upper chamber and cultured in the presence of recombinant chemokines (CXCL1 / 2/9 / 10 plus CCL2 / 5) in the lower chamber, and where, after a 24 hour incubation, the fluorescence intensity of PKH26 of the medium in the lower chamber (n = 3) was measured and expressed as the% efficacy in transwell IGNV with PKH26 +. Figure 3D includes images and a graph showing IGNV in mice with LPS-induced acute inflammatory skin disease, where DiR dye-labeled IGNVs were preincubated overnight at 4 ° C with 4T1 extract (neutralized) or without extract of 4T1 (not neutralized) before an IV injection, and where a representative image is shown (left) at 6 hours and 24 hours after the injection of each group of mice (n = 5) and followed by graphical figures (right) presented as the average net intensity (sum intensity / area, n = 5). Figure 3E includes images showing the immunohistochemical staining of chemokines (CCL2, CCL5, CXCL9 and CXCL10) expressed in human breast cancer, colon cancer tissues (lower panels), and paired adjacent tissues (upper panels), where a representative image (n = 20 for colon cancer, n = 21 for breast cancer) of each sample. Figures 3F-3G are graphs showing the results of experiments where DiR dye-labeled Ig Nv were pre-incubated at 4 ° C overnight with recombinant chemokines, as indicated in the figures, and then injected I.V. in mice with LPS-induced acute inflammatory skin disease (Figure 3F) or CT26 tumor-bearing mice (Figure 3G), where DiR dye signals were determined in skin and tumor tissues 24 hours after injection. Figure 3H is a graph showing IGNV transmigration with / without neutralization by LFA-1, where PKH26-labeled IGNVs were pre-incubated overnight with anti-LFA-1 antibody at 4 ° C and then added to the apical chamber, and where the PKH26 fluorescence intensity of the media in the lower chamber was measured after 24 hours and 48 hours of incubation and expressed as the percentage of transwell efficacy of IGNV with PKH26 +. Figure 3I includes an image and a graph showing the results of an experiment where DiR dye-labeled IGNVs were pre-incubated with antibodies against functional LFA-1 at 4 ° C overnight, washed, injected I.V. in mice with LPS-induced acute inflammatory skin disease, and DiR signals were detected after 24 hours from injection.

Figures 4A-4E include images and graphs showing targeting to human colon cancer by coating of GNV with the plasma membrane of LPS-stimulated leukocytes isolated from peripheral blood of healthy human subjects (hIGNV) or mice (mIGNV). Figures 4A-4B include graphs showing the chemokine receptor profiles of hIGNV (Figure 4A) and mIGNV (Figure 4B) based on FACS analysis, where representative histograms (n = 5) show the percentage of staining of the receptors of chemokines of hIGNV and mIGNV and where three different bands of sucrose gradients of the plasma membrane of leukocytes stimulated with LPS were used for the coating of GNV: upper band (LPS-T), middle band (LPS-M) and lower band (LPS-B). Figure 4C includes images and graphs showing the transport of DiR dye-labeled hIGNV in mice bearing SW620 colon cancer, where the mice were injected IV with DiR dye-labeled hIGNV, where live imaging of whole mice was performed. carried out on post-injection day 1 and 5, where, on post-injection day 5, tumors were removed and screened, and where DiR dye-labeled mIGNV transport was measured in a mouse model with LPS-induced acute inflammatory skin disease. Figures 4D-4E include images and a graph showing the results of an experiment where mice were injected IV with DiR dye-labeled mIGNV sin (Figure 4D) or with (Figure 4E) CXCR2 knockout, where the skin was removed. hours (Figure 4D) or 24 hours (Figure 4E) after injection and tracked. A representative image (Figures 4C-4D) of each group of mice is shown and the graphical figures are presented as the average net intensity (intensity of the sum / area, n = 5) * p <0.05, ** p < 0.01 and *** p <0.001).

Figures 5A-5F include graphs and images showing the delivery of targeted therapeutic drugs for mouse cancer and therapy for colitis. FIG. 5A is a graph showing the stability of circulating ⁱ G ⁿ V. Figure 5B includes images showing the in vitro release profile of doxorubicin from IGNV-DOX in PBS buffer with different pH values (5.0, 5.5, 6.0, 6.5 and 7.2, n = 5, ** p <0.01) . Figure 5C is a graph showing the biodistribution of doxorubicin in 4T1 tumor-bearing mice, where 4T1 tumor-bearing mice (n = 5) were injected IV with IGNV-DOX or DOX-NP®, and where doxorubicin was measured. in 4T1 tumor tissues, livers, lungs, spleens, kidneys, hearts and thymus (* p <0.05). Figure 5D includes images showing the biodistribution of doxorubicin in CT26 and 4T1 tumor tissues, where free doxorubicin (free DOX), doxorubicin supplied with GNV (GNV-DOX) and doxorubicin supplied with IGNV (IGNV-DOX) were injected IV into CT26 (n = 5) and 4T1 (n = 5) tumor-bearing mice, where tumor tissues were extracted, fixed and sectioned, and where doxorubicin was observed in tumor tissues using a confocal imaging system. Figures 5E-5F include images and graphs showing the results of experiments where CT26 and 4T1 cells were injected subcutaneously (CT26) or into a mammary fat pad (4T1) of BALB / c mice, where the mice were injected IV. with IGNV-DOX or controls, as indicated in the figure every 3 days for 30 days from 7 days after injecting tumor cells, where representative images of the tumors (Figure 5E, left panel) of each group ( n = 5), tumor volume was measured every 5 days, (figure 5E, right panel) and where the survival rate (figure 5F) of mice was calculated (* p <0.05, and ** p <0 , 01).

Figures 6A-6B include images and graphs showing chemokine receptors expressed on PMA-stimulated EL4 cells. Figure 6A includes images showing the morphology of EL4 cells cultured without PMA stimulation (left) or with PMA stimulation (right), original magnification x 10. Figure 6B includes graphs showing the percentages of chemokine receptors expressed in EL4 cells with / without PMA stimulation, where EL4 cells (n = 3) were collected at 24 hours after PMA stimulation and stained with antibodies against CCR3, CCR4, ^c C ^r 5, CCR7, CCR9, CXCR3 and Fluorescence-labeled CXCR7 and the percentage of chemokine receptors was analyzed by FACS.

Figure 7 is an image showing separation of EL4 plasma membrane through discontinuous centrifugation of the sucrose gradient, where EL4 cells with / without PMA stimulation were collected and homogenized, and where homogenized samples were in bands of sucrose and images were taken after centrifugation.

Figure 8 includes images showing the uptake of a mixture of VNG and membrane-derived vesicles, where plasma membrane vesicles derived from EL4 cells were labeled with PKH67 and VNG were labeled with PKH26, where 4T1 cells were cultured in the presence of membrane vesicles with PKH67 and PKH26-GNV mixed simply for 12 hours, and where representative images of cells (n = 3) were then taken using a confocal microscope at x 400 magnification.

Figures 9A-9B include graphs showing the stability of IGNV, where the stability of membrane-coated GNV of EL4 cells (n = 5) was analyzed at 22 ° C for a period of 5 days (Figure 9A) or at 37 ° C. for 25 hours (Figure 9B) measuring the size distribution and the surface zeta potential.

Figure 10 includes graphs showing the transmigration of GNV and IGNV, where HUVEC cells were cultured in the upper chamber coated with fibronectin (7.5 | xg / cm2) as a transmigration barrier, where GNV (upper panels) or IGNV labeled with PKH26 dye (lower panels) to the upper chamber and incubated for 24 hours and 48 hours, and where the PKH26 dye-labeled particles were measured in the middle of the lower chamber, and were expressed as the percentage of efficacy in IGNV transwells with PKH26 +.

Figure 11 includes graphs showing the expression of integrins in EL4 cells, where the EL4 cells were activated with PMA and the expression of the integrins (LFA-1 and a4p7) was detected in the cells by staining with PE antibodies against LFA-1. and a4p7 and the percentage of integrin positive cells was analyzed by FACS. Figure 12 includes graphs showing LFA-1 in I ^g N ^v , where the IGNVs were freshly prepared and conjugated with latex beads, and where the percentage of positive IGNVs in LFA-1 was quantified by Fa Cs analysis of IGNV stained with PE antibody against LFA-1.

Figures 13A-13B include images showing the purification of leukocyte plasma membranes, where leukocytes were isolated from peripheral blood of humans and mice and stimulated in vitro with (+ LPS) (100 ng / ml) or PBS as a control. (-LPS) for 24 hours, and where the homogenized plasma membranes of human cells (Figure 13a) and mouse (Figure 13B) were purified by means of gradient centrifugation of sucrose (T = upper band of the sucrose gradient purified membrane, M = middle band of the sucrose gradient purified membrane, B = lower band of the sucrose gradient purified membrane).

Figure 14 includes graphs showing chemokines in human SW620 cell culture medium, where SW620 human colorectal adenocarcinoma cells were grown in 6-well plates for 48 hours and the release of CCL2, CCL5 and CXCL10 chemokines was quantitatively analyzed in culture supernatants with an ELISA.

Figures 15A-15F include images and graphs showing the effects of IGNV on in vitro cell proliferation and potential in vivo toxicity. Figure 15A is a graph showing the results of an experiment where the proliferation of IGNV-treated 4T1 cells was analyzed using the ATPlite assay 24 hours after exposure to different concentrations of IGNV. Figure 15B is a graph showing the results of an experiment, where the number of live 4T1 cells treated with IGNV in step number 1 to 5 was determined by counting the number of live cells (no trypan blue staining). Figures 15C-15D include graphs showing the results of an experiment where TNF-α, IL-6 and IL-1 p were analyzed in serum (Figure 15C) and ALT, AST (Figure 15D) 24 hours after the mice were injected IV with IGNV three times or PBS as a control (normal control). Figure 15E includes images of H&E stained sections of livers, spleens, kidneys and lungs of mice injected with IGNV, original magnification x 20. Figure 15F includes graphs showing liver, lung, spleen, kidney and heart weights of the injected animals.

Figures 16A-16E include images and graphs showing IGNV loaded with therapeutic drugs. Figure 16A is an image showing purification of therapeutic drugs (left: doxorubicin, right: curcumin) loaded on IGNV by discontinuous sucrose density gradient centrifugation. Figures 16B-16C are graphs showing the size distribution (Figure 16B) and Zeta potential (Figure 16C) of IGNV-DOX and IGNV-Cur analyzed using a ZetaSizer. Figure 16D is a graph showing the loading efficiency of doxorubicin and curcumin detected by UV spectrometry and expressed as% = (total drugs - free drugs) / total drugs x 100%. Figure 16E includes graphs showing the release profile of doxorubicin and curcumin in PBS buffer at pH 7.2 and 37 ° C, as detected by UV spectrometry at 497 and 426 nm, respectively.

Figures 17A-17D include graphs showing the characterization of DOX-NP® and control liposomes from Avanti. Figure 17A is a graph showing the profile of doxorubicin released from dOX-NP® at 37 ° C in PBS buffer at different pH values (5.0, 5.5, 6.0, 6.5 and 7.2). Figure 17B is a graph showing the proliferation of 4T1 cells treated with different concentrations of control liposomes and analyzed using the ATPlite assay. Figures 17C-17D are graphs showing the results of an experiment where mice were injected I.V. with control liposomes daily for 3 days, where 24 hours after the last injection of the control liposomes, TNF-a, IL-6 and IL-1 p were evaluated in serum (figure 17C) and the liver enzymes AST and ALT (Figure 17D) (n = 5 mice per group).

Figure 18 includes graphs showing the biodistribution of DOX in 4T1 tumor tissues, where DOX (200 | xg) was loaded into the GNVs (GNV-DOX), then GNV-DOX was mixed or extruded with cell-derived membranes. T EL4 (IGNV-DOX), where, after washing, the GNV-DOX or IGNV-DOX were injected IV into 4T1 tumor-bearing mice, and where 24 hours later, the mice were sacrificed, tumor tissues were removed and extracted and detected ^{dO x} in tumor tissues.

Figures 19A-19E include images and graphs showing the therapeutic effect of IGNV-Cur in the inhibition of DSS-induced mouse colitis, where mice with DSS-induced colitis were injected I.V. with PBS, GNV, IGNV, free curcumin, GNV-Cur or IGNV-Cur every 2 days for 10 days, where on day 0 after the last treatment, the degree of rectal prolapse and intestinal bleeding was determined (Figure 19A), body weight was measured daily (Figure 19B), survival rates were analyzed (Figure 19C), sectioned colon tissues were stained with H&E (n = 5) (Figure 19D) and curcumin was quantified in colon tissues by HPLC (Figure 19E).

Figure 20 includes graphs showing the therapeutic effect of IGNV-Cur in the inhibition of cytokine induction of colitis tissue, where on day 3 after being administered with 2.5% DSS, the mice (n = 5 mice per group) were injected IV with PBS, free GNV (200 nmol), IGNV (200 nmol), free Cur (50 mg / kg), GNV-Cur (50 mg / kg) or IGNV-Cur (50 mg / kg), every 2 days 5 times , and where 24 hours after the last injection, TNF-a, IL-6 and IL-1 p were determined in serum by ELISA.

Figure 21 is a graph showing the determination of the micellar concentration of GNV (cmc), where different concentrations of total lipids (X-axis) extracted from grapefruit nanoparticles were used to generate a lipid film, and where, after From a sonication bath, the formation and size distribution of VNG (nm) were analyzed using a ZetaSizer.

DESCRIPTION OF EXAMPLE EMBODIMENTS

Details of one or more embodiments of the subject matter disclosed herein are set forth herein. Modifications to the embodiments described herein, and other embodiments, will be apparent to one of ordinary skill in the art after studying the information disclosed herein. The information disclosed herein, and in particular the specific details of the described embodiments, are provided primarily for the sake of clarity of understanding and no unnecessary limitations should be understood therefrom. In case of conflict, the specification herein, including definitions, will prevail.

While the terms used herein are believed to be well understood by one of ordinary skill in the art, definitions are set forth herein to facilitate explanation of the subject matter disclosed herein.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter disclosed herein belongs. Although any methods, devices, and materials similar or equivalent to those described herein may be used in the practice or testing of the subject matter disclosed herein, representative procedures, devices, and materials are described below.

Following the long-standing patent law convention, the terms "a", "an" and "the" or "the" refer to "one or one or more" when used in the present application, including the claims. Thus, for example, reference to "a cell" includes a plurality of such cells, and so on.

Unless otherwise indicated, it is to be understood that all numbers expressing ingredient amounts, properties, such as reaction conditions, and so on, used in the specification and claims, may in all cases be modified by the term "approximately". Consequently, unless otherwise indicated, the numerical parameters set forth in the present specification and claims are approximations that may vary depending on the desired properties that are sought to be obtained by the subject matter disclosed herein.

As used herein, the term "about", when referring to a value or amount of mass, weight, time, volume, concentration, or percentage, is understood to comprise variations, in some embodiments, of ± 20 %, in some embodiments, ± 10%, in some embodiments, ± 5%, in some embodiments, ± 1%, in some embodiments, ± 0.5%, and in some embodiments, ± 0.1 % of the specified amount, since these variations are appropriate to carry out the described procedure.

As used herein, ranges can be expressed as from "about" one particular value and / or to "about" another particular value. It is also understood that there are a number of values described herein and that each value is also described herein as "approximately" that particular value in addition to the value itself. For example, if the value "10" is described, then "about 10" is also described. It is also understood that each unit between two particular units is described. For example, if 10 and 15 are described, then 11, 12, 13, and 14 are also described.

The subject matter disclosed herein is based, at least in part, on the discovery that binding and coating of inflammatory cell-derived membranes into grapefruit-derived microvesicles or nanovectors (GNV) is a effective and efficient strategy to take advantage of the unlimited availability of VNG and to generate customized delivery vectors that would recognize inflammatory target sites in diseases (Figure 1A). As described herein below, such compositions have, among others, two advantages, namely: 1) the plasma membrane of activated leukocytes is preferentially bound in microvesicles formed from nanoparticles of fruit lipids; and 2) the resulting microvesicles are safe and can be used successfully for the targeted delivery of therapeutic agents to inflammatory sites.

Microvesicles are naturally occurring nanoparticles that are in the form of small assemblages of lipid particles, are about 50-1000 nm in size and are not only secreted by many types of in vitro and in vivo cell cultures, rather, they are usually found in vivo in body fluids, such as blood, urine, and malignant ascites. Indeed, microvesicles include, but are not limited to, particles, such as exosomes, epididymosomes, argosomes, exosome-like vesicles, microparticles, promininosomes, prostasomes, dexosomes, texosomes, dex, tex, archaeosomes, and oncosomes.

As noted above, microvesicles can be formed by a variety of processes, including release from apoptotic bodies, germination of microvesicles directly from the cytoplasmic membrane of a cell, and exocytosis from multivesicular bodies. For example, exosomes are usually formed by secretion from the endosomal membrane compartments of cells as a consequence of the fusion of multivesicular bodies with the plasma membrane. Multivesicular bodies are formed by germination into the endosomal membrane and subsequent strangulation of small vesicles in the luminal space. The internal vesicles present in the MVB are then released into the extracellular fluid as so-called exosomes.

As part of microvesicle formation and release, unwanted molecules are shed from cells. However, plasma membrane and cytosolic proteins are also incorporated into microvesicles during these processes, resulting in microvesicles that have particle size properties, functional properties of lipid bilayers, and other unique functional properties that allow microvesicles to function. potentially as effective nanoparticulate carriers of therapeutic agents. In this sense, the The term "microvesicle" is used herein interchangeably with the terms "nanoparticle", "liposome", "exosome", "exosome-like particle", "nano-vector" and grammatical variations of each of the foregoing. It has now been discovered, however, that edible plants, such as fruits, are not only a viable source of large numbers of microvesicles, but that edible plant-derived microvesicles can be coated with recognition cell-derived plasma membranes, such as leukocytes, and are used as an effective delivery vehicle to transport or otherwise direct microvesicles, which include any of the therapeutic agents that can be included within microvesicles, to sites of inflammation and disease.

The subject matter disclosed herein thus includes edible plant derived microvesicle compositions that are coated with recognition cell derived plasma membranes. The term "recognition cells", and grammatical variations thereof, are used herein to refer to those cells that, due to the inclusion of certain recognition moieties (eg, receptors or other proteins) in their plasma membranes They are preferably targeted or transported to certain tissues and organs in an individual's body. For example, in some embodiments, the recognition cells are activated inflammatory cells, such as leukocytes, or macrophages or neutrophils that preferentially recognize or target sites of inflammation. As another example, in some embodiments, the target cells are circulating tumor cells or metastatic tumor cells that preferentially target tissues, such as brain, lung, liver, and bone tissue (ie, frequent sites of metastasis). . As yet another example, in some embodiments, the recognition cells are stem cells (eg, stem cells derived from bone marrow) that, preferably, can be targeted and used for tissue regeneration.

In some embodiments, the microvesicle compositions further include therapeutic agents and are useful in the treatment of various diseases, including inflammatory disorders and cancers. In some embodiments of the matter disclosed herein, a microvesicle composition is disclosed comprising a microvesicle derived from an edible plant and a plasma membrane covering the microvesicle, wherein the plasma membrane has been derived from a cell. of recognition. In some embodiments, a composition is disclosed in which a therapeutic agent is encapsulated by the microvesicle derived from the edible plant. In some embodiments, the therapeutic agent encapsulated by the edible plant derived microvesicle is selected from a phytochemical agent and a chemotherapeutic agent.

The term "edible plant" is used herein to describe organisms of the kingdom Plantae that are capable of producing their own food, at least in part, from inorganic matter through photosynthesis, and that are suitable for consumption by an individual, as defined herein, below. Such edible plants include, but are not limited to, vegetables, fruits, nuts, and the like. In some embodiments of the microvesicle compositions described herein, the edible plant is a fruit. In some embodiments, the fruit is selected from a grape, a grapefruit, and a tomato.

The phrase "derived from an edible plant", when used in the context of a microvesicle derived from an edible plant, refers to a microvesicle which, through human hands, exists separate from its native environment and therefore it is not a product of nature. In this regard, in some embodiments, the term "derived from an edible plant" may be used interchangeably with the phrase "isolated from an edible plant" to describe a microvesicle of the matter disclosed herein that is useful for coating. with a plasma membrane derived from inflammatory cells and / or for encapsulation of therapeutic agents.

The phrase "encapsulated by a microvesicle", or grammatical variations thereof, is used herein to refer to microvesicles whose lipid bilayer surrounds a therapeutic agent. For example, a reference to "microvesicle curcumin" refers to a microvesicle whose lipid bilayer encapsulates or surrounds an effective amount of curcumin. In some embodiments, encapsulation of various therapeutic agents within microvesicles can be achieved by first mixing the one or more of the phytochemicals or chemotherapeutic agents with isolated microvesicles in a suitable buffered solution, such as phosphate buffered saline (PBS). After a sufficient incubation period to allow the therapeutic agent to be encapsulated during the incubation period, the microvesicle / therapeutic agent mixture is then subjected to a sucrose gradient (e.g., 8,30 sucrose gradient , 45 and 60%) to separate the free therapeutic agent and free microvesicles from the therapeutic agents encapsulated within the microvesicles, and a centrifugation step to isolate the microvesicles that encapsulate the therapeutic agents. After this centrifugation step, the microvesicles that include the therapeutic agents are observed as a band in the sucrose gradient, so that they can then be collected, washed and dissolved in a solution suitable for use, as shown. described in this document, below.

As indicated, in some embodiments, the therapeutic agent is a phytochemical agent. As used herein, the term "phytochemical agent" refers to a non-nutritive plant-derived compound, or an analog thereof. Examples of phytochemicals include, but are not limited to, compounds, such as monophenols; flavonoids, such as flavonols, flavanones, flavones, flavan-3-ols, anthocyanins, anthocyanidins, isoflavones, dihydroflavonols, chalcones and coumestanes; phenolic acids; hydroxycinnamic acids; lignans; tyrosol esters; stilbenoids; hydrolyzable tannins; carotenoids, such as carotenes and xanthophylls; monoterpenes; saponins; lipids, such as phytosterols, tocopherols, and omega-3,6,9 fatty acids; diterpenes; triterpinoids; betalains, such as betacyanins and betaxanthins; dithiolthions; thiosulfonates; indoles; and glucosinolates. As another example of a phytochemical agent described herein, the phytochemical agent may be an analog of a plant-derived compound, such as oltipraz, which is an analog of 1,2-dithiol-3-thione, a compound that is found in many cruciferous vegetables.

In some embodiments of the matter disclosed herein, the therapeutic agent is a phytochemical agent selected from curcumin, resveratrol, baicalein, fisetin, and quercetin. In some embodiments, the phytochemical is curcumin. Curcumin is a natural pleiotropic polyphenol with anti-inflammatory, antineoplastic, antioxidant and chemopreventive activity, these activities having been identified both at the protein and molecular level. However, limited progress has been reported with regard to the therapeutic use of curcumin, as curcumin is insoluble in aqueous solvents and is relatively unstable. Furthermore, curcumin is known to have low systemic bioavailability after oral dosing, further limiting its use and clinical efficacy. It has been determined, however, that by encapsulating curcumin in microvesicles derived from edible plants, not only can the solubility of curcumin be increased, but encapsulation of curcumin within microvesicles protects curcumin from degradation and it also increases the bioavailability of curcumin in the microvesicle.

As also noted hereinbefore, in some embodiments of the matter disclosed herein, the therapeutic agent that is encapsulated within the exosome is a chemotherapeutic agent. Examples of chemotherapeutic agents that may be used, depending on the subject matter disclosed herein, include, but are not limited to, platinum coordination compounds, such as cisplatin, carboplatin, or oxaliplatin; taxane compounds, such as paclitaxel or docetaxel; topoisomerase I inhibitors, such as camptothecin compounds, for example irinotecan or topotecan; topoisomerase II inhibitors, such as podophyllotoxin antitumor derivatives, eg, etoposide or teniposide; antitumor vinca alkaloids, for example vinblastine, vincristine or vinorelbine; derivatives of antitumor nucleosides, eg, 5-fluorouracil, gemcitabine or capecitabine; alkylating agents, such as nitrogen mustard or nitrosourea, for example cyclophosphamide, chlorambucil, carmustine or lomustine; antitumor anthracycline derivatives, for example daunorubicin, doxorubicin, idarubicin or mitoxantrone; HER2 antibodies, eg, trastuzumab; estrogen receptor antagonists or selective estrogen receptor modulators, eg, tamoxifen, toremifene, droloxifene, faslodex, or raloxifene; aromatase inhibitors, such as exemestane, anastrozole, letrazole, and vorozole; differentiating agents, such as retinoids and retinoic acid metabolism blocking agents (RAMBA), eg, accutane; DNA methyl transferase inhibitors, eg, azacytidine; kinase inhibitors, eg, flavoperidol, imatinib mesylate, or gefitinib; farnesyltransferase inhibitors; HDAC inhibitors; other inhibitors of the ubiquitin-proteasome pathway, eg, VELCADE® (Millennium Pharmaceuticals, Cambridge, MA); or YONDELIS® (Johnson & Johnson, New Brunswick, NJ). In some embodiments, the chemotherapeutic agent that is encapsulated by an exosome, according to the subject matter disclosed herein, is selected from retinoic acid, 5-fluorouracil, vincristine, actinomycin D, adriamycin, cisplatin, docetaxel, doxorubicin, and taxol.

In other embodiments of the art disclosed herein, therapeutic agents included within microvesicle compositions comprise nucleic acid molecules selected from a siRNA, a microRNA, and an expression vector, such as a mammalian expression vector. The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single or double stranded form. Unless specifically limited, the term encompasses nucleic acids that contain known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (eg, degenerate codon substitutions) and complementary sequences, as well as the explicitly stated sequence. Specifically, degenerate codon substitutions can be achieved through sequence generation, in which the third position of one or more selected codons (or all) is replaced by mixed base residues and / or deoxyinosine residues (Batzer et al. al. (1991) Nucleic Acid Res 19: 5-081; Ohtsuka et al. (1985) J Biol Chem. 260: 2605-2608; Rossolini et al (1994) Mol Cell Probes 8: 91-98). The terms "nucleic acid" or "nucleic acid sequence" can also be used interchangeably with gene, open reading frame (ORF), cDNA, mRNA, siRNA, microRNA, and the like.

The terms "small interference RNA", "short interference RNA", "small hairpin RNA", "siRNA", and "hRNA" are used interchangeably herein to refer to any nucleic acid molecule capable of mediating in RNA interference (RNAi) or gene silencing. See, for example, Bass, Nature 411: 428-429, 2001; Elbashir et al., Nature 411: 494-498, 2001a; and patents WO 00/44895, W ^or 01/36646, WO 99/32619, WO 00/01846, WO 01/29058, WO 99/07409 and WO 00/44914. In one embodiment, the siRNA may comprise a double-stranded polynucleotide molecule comprising complementary sense and antisense regions, wherein the antisense region comprises a sequence complementary to a region of a target nucleic acid molecule. In another embodiment, the siRNA may comprise a single-stranded polynucleotide having sense and antisense self-complementary regions, wherein the antisense region comprises a sequence complementary to a region of a target nucleic acid molecule. In yet another embodiment, the siRNA may comprise a single stranded polynucleotide having one or more loop structures and a stem comprising sense and antisense self-complementary regions, wherein the antisense region comprises a sequence complementary to a region of a molecule. of target nucleic acid, and wherein the polynucleotide can be processed in vivo or in vitro to generate an active siRNA capable of mediating RNAi. As used herein, siRNA molecules need not be limited to those molecules that contain only RNA, but also encompass chemically modified nucleotides and non-nucleotides.

MicroRNAs are small, naturally occurring, noncoding RNAs that are from about 17 to about 25 nucleotide bases (nt) in length in their biologically active form. The miRNAs post-transcription regulate gene expression by repressing the translation of the target mRNA. It is believed that miRNAs function as negative regulators, that is, higher amounts of a specific miRNA will correlate with lower levels of expression of the target gene. There are three forms of miRNA that exist in vivo, primary miRNA (priRNA), premature miRNA (preRNA) and mature miRNA. Primary miRNAs (priRNAs) are expressed as stem-loop structured transcripts of approximately a few hundred bases to greater than 1 kb. The priRNAmi transcripts are cleaved in the nucleus by an RNase II endonuclease called Drosha that cleaves both strands of the stem near the base of the stem loop. Drosha cleaves the RNA duplex with staggered cuts, leaving a 5'phosphate and 2 nt overhang at the 3 'end. The cleavage product, premature mRNA (mi preRNA) is about 60 to about 110 nt in length with a hairpin structure formed in a reverse-folded shape. The preRNAmi is transported from the nucleus to the cytoplasm by Ran-GTP and Exportin-5. The preRNAs are further processed in the cytoplasm by another RNase II endonuclease called Dicer. Dicer recognizes the 5 'phosphate and the 3' overhang, and cleaves the loop at the stem-loop junction to form miRNA duplexes. The miRNA duplex binds to the RNA-induced silencing complex (RISC), where the antisense strand is preferentially degraded and the mature sense strand miRNA directs the RISC to its target site. It is the mature miRNA that is the biologically active form of miRNA and is about 17 to about 25 nt in length.

In some embodiments, nucleic acid molecules that are encapsulated or, in any event, incorporated in a microvesicle composition of matter disclosed herein that are included in the microvesicles are part of an expression vector. The term "expression vector" is used interchangeably herein with the terms "expression cassette" and "expression control sequence", and is used to refer to a nucleic acid molecule capable of directing the expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to the nucleotide sequence of interest that is operably linked to termination signals. It also usually comprises sequences required for proper translation of the nucleotide sequence. The coding region usually encodes a polypeptide of interest, but may also encode a functional RNA of interest, for example, antisense RNA or an untranslated, sense or antisense RNA, or a non-coding RNA (cRNA), such as a small or long cRNA. The expression vector comprising the nucleotide sequence of interest can be chimeric, which means that at least one of its components is heterologous with respect to at least one of its other components. The expression vector can also be one that is naturally occurring, but has been obtained in a recombinant form useful for heterologous expression. In some embodiments, the expression vector is a mammalian expression vector that is capable of directing the expression of a particular nucleic acid sequence of interest in a mammalian cell.

With respect to the plasma membrane that lines the microvesicles derived from edible plants, the phrase "that lines the microvesicle", and variations thereof, is used herein to refer to the coating, placement and / or attachment of a plasma membrane. to the lipid bilayer of an exemplary microvesicle of the subject matter disclosed herein, or a portion thereof. In some embodiments, coating of a microvesicle with a plasma membrane derived from an activated inflammatory cell, such as a leukocyte, is achieved by initial exposure of a population of inflammatory cells to a cell activating compound (eg, phorbol 12 - myristate 13-acetate (PMA), a calcium ionophore, such as ionomycin, or a protein transport inhibitor) capable of inducing the expression of various cytokines, chemokines, and / or chemokine receptors. After a period of incubation of the inflammatory cells with said activating compound, the inflammatory cells are then lysed and homogenized, and the plasma membranes are collected by sucrose density gradient centrifugation. After collecting the plasma membranes, the membranes are subsequently sonicated to form vesicles. Such vesicles can then be combined with the microvesicles described herein (eg, microvesicles encapsulating a therapeutic agent) and coextruded through a membrane to thereby coat the microvesicles with the derived plasma membranes. of inflammatory cells. In this regard, and without wishing to be bound by any particular theory, it is believed that, due to the Using plasma membranes derived from activated inflammatory cells, the resulting microvesicle compositions include a plasma membrane coating that has one or more chemokine receptors or other recognition moieties that are capable of transporting or, in any event, directing the compositions to the site. of inflamed or diseased tissue.

In some embodiments of the matter disclosed herein, a pharmaceutical composition is disclosed comprising an edible plant-derived microvesicle composition described herein and a pharmaceutical vehicle, carrier, or excipient. In some embodiments, the pharmaceutical composition is pharmaceutically acceptable in humans. Furthermore, as further described below, in some embodiments, the pharmaceutical composition can be formulated as a therapeutic composition for delivery to an individual.

A pharmaceutical composition, as described herein, preferably comprises a composition that includes a pharmaceutical carrier, such as aqueous and non-aqueous sterile injection solutions that may contain antioxidants, buffers, bacteriostats, bactericidal antibiotics, and solutes. which make the formulation isotonic with the body fluids of the intended recipient; and sterile aqueous and non-aqueous suspensions, which may include suspending agents and thickening agents. The pharmaceutical compositions used can take forms, such as suspensions, solutions or emulsions, in oily or aqueous vehicles, and can contain formulating agents, such as suspending, stabilizing and / or dispersing agents. Additionally, the formulations can be presented in unit dose or multiple dose containers, for example, sealed ampoules and vials, and can be stored in a frozen or lyophilized state or at room temperature (lyophilized) requiring only the addition of the sterile liquid carrier immediately prior to the utilization.

In some embodiments, solid formulations of compositions for oral administration may contain suitable carriers or excipients, such as corn starch, gelatin, lactose, acacia, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, calcium carbonate, calcium chloride. sodium or alginic acid. Disintegrators that can be used include, but are not limited to, microcrystalline cellulose, corn starch, sodium starch glycolate, and alginic acid. Tablet binders that can be used include acacia, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, hydroxypropyl methylcellulose, sucrose, starch, and ethylcellulose. Lubricants that can be used include magnesium stearates, stearic acid, silicone fluid, talc, waxes, oils, and colloidal silica. In addition, solid formulations can be uncoated or can be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thus provide sustained / extended action over a longer period of time. For example, glyceryl monostearate or glyceryl distearate can be used to provide a sustained / extended release formulation. A person skilled in the art knows of numerous techniques for the formulation of sustained release preparations and can be used, according to the present invention, including the techniques described in the following references: US Patents 4,891,223; 6,004,582; 5,397,574; 5,419,917; 5,458,005; 5,458,887; 5,458,888; 5,472,708; 6,106,862; 6,103,263; 6,099,862; 6,099,859; 6,096,340; 6,077,541; 5,916,595; 5,837,379; 5,834,023; 5,885,616; 5,456,921; 5,603,956; 5,512,297; 5,399,362; 5,399,359; 5,399,358; 5,725,883; 5,773,025; 6,110,498; 5,952,004; 5,912,013; 5,897,876; 5,824,638; 5,464,633; 5,422,123; and 4,839,177 and WO 98/47491.

Liquid preparations for oral administration can take the form, for example, of solutions, syrups or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use. Said liquid preparations can be prepared by conventional techniques with pharmaceutically acceptable additives, such as suspending agents (for example, sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (eg, lecithin or acacia); non-aqueous vehicles (eg, almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (eg, methyl or propyl p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavorings, colors, and sweeteners, as appropriate. Preparations for oral administration can be suitably formulated to provide a controlled release of the active compound. For buccal administration, the compositions may take the form of capsules, tablets or lozenges formulated in a conventional manner.

Various liquid and powder formulations can also be prepared by conventional procedures for inhalation into the lungs of the individual to be treated or for intranasal administration into the nose and sinus cavities of an individual to be treated. For example, the compositions can be conveniently delivered in the form of an aerosol spray presentation from pressurized containers or a nebulizer, using a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. Capsules and cartridges, for example gelatin for use in an inhaler or insufflator, can be formulated containing a powder mixture of the desired compound and a suitable powder base, such as lactose or starch.

The compositions can also be formulated as a preparation for implantation or injection. Thus, for example, the compositions can be formulated with suitable polymeric or hydrophobic materials (e.g. for example, as an emulsion in an acceptable oil) or ion exchange resins, or as poorly soluble derivatives (for example, as a poorly soluble salt).

Injectable formulations of the compositions can contain various carriers, such as vegetable oils, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, polyols (glycerol, propylene glycol, liquid polyethylene glycol), and the like. For intravenous injections, water-soluble versions of the compositions can be administered by the drip procedure, whereby, a formulation is infused that includes a pharmaceutical composition of matter disclosed herein and a physiologically acceptable excipient. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipients. Intramuscular preparations, for example, a sterile formulation of a suitable soluble salt form of the compounds, can be dissolved and administered in a pharmaceutical excipient, such as water for injection, 0.9% saline, or 5-glucose solution. %. A suitable insoluble form of the composition may be prepared and administered as a suspension in an aqueous base or a pharmaceutically acceptable oil base, such as an ester of a long chain fatty acid (eg, ethyl oleate).

In addition to the formulations described above, the microvesicle compositions of matter disclosed herein can also be formulated as rectal compositions, such as suppositories or retention enemas, for example, containing conventional suppository bases, such as cocoa butter. or other glycerides. In addition, the exosomal compositions can also be formulated as a depot preparation by combining the compositions with suitable polymeric or hydrophobic materials (eg, as an emulsion in an acceptable oil) or ion exchange resins, or as poorly soluble derivatives, for example, as a poorly soluble salt.

In some embodiments of the subject matter disclosed herein, a composition is further disclosed, as described herein, for use in treating an inflammatory disorder or cancer. In some embodiments, treating an inflammatory disorder comprises administering to an individual in need thereof an effective amount of a microvesicle composition of matter disclosed herein.

As used herein, the terms "treatment" or "treat" refer to any treatment of a condition of interest (eg, an inflammatory disorder or a cancer), including, but not limited to, prophylactic treatment and therapeutic treatment. Therefore, the terms "treatment" or "treat" include, but are not limited to: preventing a condition of interest or the development of a condition of interest; inhibit the progression of a condition of interest; stop or prevent the further development of a condition of concern; reduce the severity of a condition of concern; improve or alleviate symptoms associated with a condition of concern; and causing a regression of a condition of interest or of one or more of the symptoms associated with a condition of interest.

As used herein, the term "inflammatory disorder" includes diseases or disorders that are caused, at least, in part, or exacerbated, by inflammation, which, in general, is characterized by increased blood flow, edema, activation of immune cells (eg, proliferation, cytokine production, or increased phagocytosis), warmth, redness, swelling, pain, and / or loss of function in the affected tissue or organ. The cause of inflammation can be due to physical damage, chemicals, microorganisms, tissue necrosis, cancer, or other agents or conditions.

Inflammatory disorders include acute inflammatory disorders, chronic inflammatory disorders, and recurrent inflammatory disorders. Acute inflammatory disorders, in general, are of relatively short duration, lasting from about a few minutes to about one or two days, although they can last for several weeks. Features of acute inflammatory disorders include increased blood flow, exudation of fluid and plasma proteins (edema), and emigration of leukocytes, such as neutrophils. Chronic inflammatory disorders, in general, are of longer duration, eg, weeks to months to years or more, and are associated histologically with the presence of lymphocytes and macrophages and with proliferation of blood vessels and connective tissue. Recurrent inflammatory disorders include disorders that recur over a period of time or have periodic episodes. Some inflammatory disorders fall into one or more categories. Exemplary inflammatory disorders include, but are not limited to: atherosclerosis; arthritis; cancers caused by inflammation; asthma; autoimmune uveitis; adoptive immune response; dermatitis; multiple sclerosis; complications of diabetes; osteoporosis; Alzheimer disease; cerebral malaria; hemorrhagic fever; autoimmune disorders; and inflammatory bowel disease. In some embodiments, the inflammatory disorder is selected from the group comprising sepsis, septic shock, colitis, colon cancer, and arthritis.

For the administration of a therapeutic composition, as described herein (e.g., a microvesicle derived from edible plants that encapsulates a therapeutic agent), conventional procedures of extrapolation of human dosage based on administered doses can be carried out. to a murine animal model using the conversion factor for conversion from the mouse dosage to the human dosage: human dose per kg = mouse dose per kg x 12 (Freireich, et al., (1966) Cancer Chemother Rep. 50: 219-244). Doses can also be given in milligrams per square meter of body surface area because this procedure, rather than body weight, correlates well with certain metabolic and excretory functions. Additionally, body surface area can be used as a common denominator for drug dosing in adults and children, as well as in different animal species, as described by Freireich, et al. (Freireich et al., (1966) Cancer Chemother Rep. 50: 219-244). Briefly, to express a dose in mg / kg in any given species as the equivalent dose in mg / m2, multiply the dose by the appropriate km factor. In an adult human, 100 mg / kg is equivalent to 100 mg / kg x 37 kg / m2 = 3,700 mg / m2.

Suitable methods for the administration of a therapeutic composition, according to the methods of matter disclosed herein, include, but are not limited to, systemic administration, parenteral administration (including intravascular, intramuscular, and / or intraarterial), oral delivery, buccal delivery, rectal delivery, subcutaneous administration, intraperitoneal administration, inhalation, intratracheal instillation, surgical implantation, transdermal delivery, local injection, intranasal delivery, and hypervelocity injection / bombardment. Where applicable, continuous infusion can enhance drug accumulation at a target site (see, for example, US Patent 6,180,082).

Regardless of the route of administration, the compositions of matter disclosed herein are usually administered in an amount effective to achieve the desired response. Thus, the term "effective amount" is used herein to refer to an amount of the therapeutic composition (eg, a microvesicle encapsulating a therapeutic agent and a pharmaceutical vehicle, carrier, or excipient) sufficient to elicit a biological response. measurable (for example, a decrease in inflammation). Actual dosage levels of the active ingredients in a therapeutic composition of the present invention can be varied in order to administer an amount of the active compound or compounds that is effective to achieve the desired therapeutic response for a particular individual and / or application. Of course, the effective amount in any particular case will depend on a variety of factors, including the activity of the therapeutic composition, the formulation, the route of administration, the combination with other drugs or treatments, the severity of the condition to be treated, and the physical condition and prior medical history of the individual being treated. Preferably, a minimal dose is administered and the dose is scaled in the absence of dose-limiting toxicity to a minimally effective amount. Determining and adjusting a therapeutically effective dose, as well as evaluating when and how to make such adjustments, are known to one of ordinary skill in the art.

For additional guidance regarding formulation and dosage, see US Patents 5,326,902; 5,234,933; PCT International Publication No. WO 93/25521; Berkow et al., (1997) The Merck Manual of Medical Information, Home ed. Merck Research Laboratories, Whitehouse Station, NJ; Goodman et al., (1996) Goodman & Gilman's the Pharmacological Basis of Therapeutics, 9th ed. McGraw-Hill Professions Division, New York; Ebadi, (1998) CRC Desk Reference of Clinical Pharmacology. CRC Press, Boca Raton, Florida; Katzung, (2001) Basic & Clinical Pharmacology, 8th ed. Lange Medical Books / McGraw-Hill Medical Pub. Division, New York; Remington et al., (1975) Remington's Pharmaceutical Sciences, 15th ed. Mack Pub. Co., Easton, Pennsylvania; and Speight et al, (1997) Avery's Drug Treatment: A Guide to the Properties, Choice, Therapeutic Use and Economic Value of Drugs in Disease Management, 4th ed. Adis International, Auckland / Philadelphia; Duch et al., (1998) Toxicol. Lett. 100-101: 255-263.

In some embodiments of the therapeutic methods disclosed herein, administration of an edible plant-derived microvesicle composition of the matter disclosed herein reduces the amount of an inflammatory cytokine in an individual. In some embodiments, the inflammatory cytokine can be interleukin-1 p (IL-1P), tumor necrosis factor alpha (TNF-a), interferon-y (IFN-y), or interleukin-6 (IL-6).

Various procedures known to one skilled in the art can be used to determine the reduction in the amount of inflammatory cytokines in an individual. For example, in certain embodiments, the amount of expression of an inflammatory cytokine in an individual can be determined by probing for mRNA of the gene encoding the inflammatory cytokine in a biological sample obtained from the individual (e.g., a tissue sample, a sample urine, a saliva sample, a blood sample, a serum sample, a plasma sample or sub-fractions thereof) using any RNA identification assay known to a person skilled in the art. Briefly, RNA can be extracted from the sample, amplified, converted to cDNA, labeled and allowed to hybridize with probes of a known sequence, such as known RNA hybridization probes immobilized on a substrate, for example, an array or microarray, or is quantified by real-time PCR (eg, quantitative real-time PCR, as available from Bio-Rad Laboratories, Hercules, CA). Because the probes to which the nucleic acid molecules in the sample bind are known, the molecules in the sample can be identified. In this regard, DNA probes for one or more of the mRNAs encoded by inflammatory genes can be immobilized on a substrate and are provided for use in the practice of a method, according to the subject matter disclosed herein.

With regard to additionally determining inflammatory cytokine levels in samples, mass spectrometry and / or immunoassay devices and procedures can also be used to measure inflammatory cytokines in samples, although other procedures can also be used and are well known to a person skilled in the art. See, for example, US Patents 6,143,576; 6,113,855; 6,019,944; 5,985,579; 5,947,124; 5,939,272; 5,922,615; 5,885,527; 5,851,776; 5,824,799; 5,679,526; 5,525,524 and 5,480,792. Immunoassay devices and procedures can utilize labeled molecules in various competitive or non-competitive sandwich assay formats to generate a signal that is related to the presence or amount of an analyte of interest. In addition, certain methods and devices, such as optical biosensors and immunoassays, can be used to determine the presence or quantity of analytes without the need for a labeled molecule. See, for example, US Patents 5,631,171 and 5,955,377.

Any suitable immunoassay may be used, for example, enzyme-linked immunoassays (ELISA), radioimmunoassays (RIA), competitive binding assays, and the like. Specific immunological binding of the antibody to the inflammatory molecule can be detected directly or indirectly. Direct labels include fluorescent or luminescent labels, metals, dyes, radionucleotides, and the like, attached to the antibody. Indirect markers include various enzymes well known in the art, such as alkaline phosphatase, horseradish peroxidase, and the like.

The use of immobilized antibodies or fragments thereof specific for inflammatory molecules is also contemplated in the present invention. Antibodies can be immobilized on a variety of solid supports, such as magnetic particles or chromatographic matrix particles, the surface of an assay plate (such as microtiter wells), pieces of a solid substrate material (such as plastic, nylon, paper) and the like. A test strip can be prepared by coating the antibody or a plurality of antibodies in a matrix on a solid support. This strip can then be dipped into the biological test sample and then rapidly processed through washes and detection steps to generate a measurable signal, such as, for example, a colored spot.

Mass spectrometry (MS) analysis can be used, either alone or in combination, with other procedures (eg, immunoassays), to determine the presence and / or amount of an inflammatory molecule in an individual. Exemplary MS analyzes that may be used in accordance with the present invention include, but are not limited to: liquid chromatography-mass spectrometry (LC-MS); matrix-assisted laser desorption / ionization (MALDI-TOF-Ms) time-of-flight mass spectrometry analysis, such as MALDI-TOF direct-spot or MALDI-TOF mass spectrometry analysis with chromatography liquid; electrospray ionization mass spectrometry (ESI-MS), such as, for example, ESI-MS with liquid chromatography (LC); and Surface Enhanced Laser Desorption / Ionization Mass Spectrometry Analysis (SELDI-TOF-MS). Each of these types of mass spectrometric analysis can be carried out using commercially available spectrometers, such as, for example, triple quadrupole mass spectrometers. Procedures for using mass spectrometric analysis to detect the presence and amount of peptides, such as inflammatory cytokines, in biological samples are known in the art. See, for example, US Patents 6,925,389; 6,989,100 and 6,890,763.

With further regard to the various therapeutic procedures described herein, although certain embodiments of the procedures described herein require only a qualitative evaluation (eg, the presence or absence of expression of an inflammatory cytokine in an individual), other embodiments of the procedures require a quantitative evaluation (eg, the amount of increase in the level of an inflammatory cytokine in an individual). Such quantitative evaluations can be performed, for example, using one of the above-mentioned procedures, as will be understood by one skilled in the art.

The person skilled in the art will also understand that the measurement of a reduction in the amount of a certain characteristic (eg cytokine levels) or an improvement in a certain characteristic (eg inflammation) in an individual is a statistical analysis. For example, a reduction in an amount of inflammatory cytokines in an individual can be compared to the control level of inflammatory cytokines and an amount of inflammatory cytokines less than or equal to the control level can be indicative of a reduction in the amount of inflammatory cytokines. , as evidenced by a level of statistical significance. Statistical significance is often determined by comparing two or more populations, and determining a confidence interval and / or a p-value. See, for example, Dowdy and Weardon, Statistics for Research, John Wiley & Sons, New York, 1983. Preferred confidence intervals for the present subject are 90%, 95%, 97.5%, 98%, 99% , 99.5%, 99.9% and 99.99%, while the preferred p-values are 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001 and 0.0001.

In some embodiments, a composition, as described herein, is further disclosed for use in treating cancer. In some embodiments, treating a cancer comprises administering to an individual in need thereof an effective amount of an edible plant-derived microvesicle composition of the matter disclosed herein (i.e., when a plasma membrane-coated microvesicle encapsulates a therapeutic agent). In some embodiments, the therapeutic agent encapsulated within the plasma membrane-coated microvesicle and used to treat cancer is selected from a phytochemical agent and a chemotherapeutic agent, as such agents have been found to be particularly useful in treating cancer. Cancer. As used herein, the term "cancer" refers to all types of cancer or neoplasm or malignant tumors found in animals, including leukemias, carcinomas, melanoma, and sarcomas.

By "leukemia" is meant widely progressive malignant diseases of the blood-forming organs and is generally characterized by distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Leukemia diseases include, for example, acute non-lymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocytemic leukemia, basophilic leukemia, leukemia basal cell leukemia, bovine leukemia, chronic myelocytic leukemia, cutaneous leukemia, embryonal leukemia, eosinophilic leukemia, Gross leukemia, hairy cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, leukemia of the stem cell, leukemia of monocopy, acute leukemia of stem cells lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenic leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myeloid leukemia, granulocytic leukemia, myeloid leukemia, granulocytic leukemia Naegeli leukemia, plasma cell leukemia, plasmacytic leukemia, promyelocytic leukemia, Rieder cell leukemia, Schilling leukemia, stem cell leukemia, subleukemic leukemia, and undifferentiated cell leukemia.

The term "carcinoma" refers to a new malignant growth composed of epithelial cells that tend to infiltrate the surrounding tissues and metastasize. Examples of carcinomas include, for example, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, adenomatous carcinoma, carcinoma of the adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, basal cell carcinoma, basaloid, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comed carcinoma, corpus carcinoma, cylindrical carcinoma, cutaneous carcinoma, carcinoma of the skin cylindrical, duct carcinoma, hard carcinoma, embryonal carcinoma, encephaloid carcinoma, epienoid carcinoma, adenoid epithelial carcinoma, exophytic carcinoma, ex ulcere carcinoma, fibrous carcinoma, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, giant cell carcinoma glandular carcinoma, granulosa cell carcinoma, hairy matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypemephroid carcinoma, childhood embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, Kromcher intraepithelial carcinoma, intraepithelial carcinoma , Kulchitzky cell carcinoma, large cell carcinoma, lenticular carcinoma, lenticular carcinoma, lipomatous carcinoma, lymphoepithelial carcinoma, medullary carcinoma, medullary carcinoma, melanotic carcinoma, molle carcinoma, mucinous carcinoma, muciparum carcinoma, mucoepithelial carcinoma, mucoepithelial carcinoma, mucoepithelial carcinoma, mucoepithelial carcinoma mucosal carcinoma, myxomatous carcinoma, nasopharyngeal carcinoma, oat cell carcinoma, ossifying carcinoma, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, spiny cell carcinoma, pultacin cell carcinoma, renal cell carcinoma of the kidneyreserve, sarcomatoid carcinoma, Schneiderian carcinoma, scirrous carcinoma, scrotal carcinoma, signet ring cell carcinoma, simplex carcinoma, small cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, spongy carcinoma, carcinoma squamous cell carcinoma, squamous cell carcinoma, chain carcinoma, telangiectatic carcinoma, telangiectodes carcinoma, transitional cell carcinoma, tuberosum carcinoma, tuberous carcinoma, warty carcinoma and villosum carcinoma.

The term "sarcoma" refers, in general, to a tumor that is composed of a substance similar to embryonic connective tissue and, in general, is composed of tightly packed cells embedded in a fibrillar or homogeneous substance. Sarcomas include, for example, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, soft part alveolar sarcoma, ameloblastic sarcoma, botryoid sarcoma, embryonic sarcoma, chorio carcinoma, sarcoma , Wilns tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, B-cell immunoblastic sarcoma, lymphoma, Immunoblastic T-cell sarcoma, Jensen's sarcoma, Kaposi's sarcoma, Kupffer's cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymal sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, and telangiectaltic sarcoma.

The term "melanoma" is understood to mean a tumor arising from the melanocytic system of the skin and other organs. Melanomas include, for example, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanone, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungual melanoma, and superficial spreading melanoma.

Additional cancers include, for example, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small cell lung tumors , primary brain tumors, stomach cancer, colon cancer, malignant pancreatic insulanoma, malignant carcinoid, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cancer cervical, endometrial cancer and adrenal cortical cancer. In some embodiments, the cancer is selected from the group comprising skin cancer, head and neck cancer, colon cancer, breast cancer, brain cancer, and lung cancer.

As used herein, the term "individual" includes human and animal individuals. Thus, veterinary therapeutic uses are disclosed, according to the subject matter disclosed herein. Therefore, the subject matter disclosed in the present document discloses the treatment of mammals, such as humans, as well as those mammals of importance because they are in danger of extinction, such as the Siberian tigers; of economic importance, such as animals raised on farms for consumption by humans; and / or animals of social importance to humans, such as animals kept as pets or in zoos. Examples of such animals include, but are not limited to: carnivores, such as cats and dogs; swine, including pigs, hogs, and wild boar; ruminants and / or ungulates, such as cows, oxen, sheep, giraffes, deer, goats, bison, and camels; and horses. The treatment of birds is also disclosed, which includes the treatment of those types of birds that are in danger of extinction and / or are kept in zoos, as well as poultry and, more particularly domesticated poultry, that is, farm birds, such as turkeys, chickens, ducks, geese, guinea fowl, and the like, as they are also of economic importance to humans. Therefore, the treatment of livestock is also disclosed, including, but not limited to, domesticated pigs, ruminants, ungulates, horses (including racehorses), farm birds, and the like.

The practice of the matter disclosed herein may utilize, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the ability of technique. These techniques are fully explained in the literature. See, for example, Molecular Cloning A Laboratory Manual (1989), 2nd Ed., Ed. By Sambrook, Fritsch and Maniatis, editors, Cold Spring Harbor Laboratory Press, chapters 16 and 17; US Patent 4,683,195; DNA Cloning, Volumes I and II, Glover, ed., 1985; Oligonucleotide Synthesis, M.J. Gait, ed., 1984; Nucleic Acid Hybridization, D. Hames and S.J. Higgins, editors, 1984; Transcription and Translation, B.D. Hames and S.J. Higgins, editors, 1984; Culture of Animal Cells, R.I. Freshney, Alan R. Liss, Inc., 1987; Immobilized Cells and Enzymes, IRL Press, 1986; Perbal (1984), A Practical Guide To Molecular Cloning; see Methods in Enzymology (Academic Press, Inc., New York); Gene Transfer Vectors for Mammalian Cells, J.H. Miller and M. P. Calos, editors, Cold Spring Harbor Laboratory, 1987; Methods in Enzymology, Volumes 154 and 155, Wu et al., Editors, Academic Press Inc., NY; Immunochemical Methods in Cell and Molecular Biology (Mayer and Walker, editors, Academic Press, London, 1987 ;. Handbook of Experimental Immunology, volumes I-IV, DM Weir and CC Blackwell, editors, 1986. The subject matter disclosed herein is further illustrated by the following specific, but not limiting, examples.

EXAMPLES

Inflammation is a hallmark of many diseases. Activated immune cells are intrinsically capable of targeting inflammatory sites. Using three mouse models of inflammation-induced disease, the following studies demonstrated that grapefruit-derived nanovectors (GNV) coated with activated leukocyte membranes enriched with inflammatory chemokine receptor (IGNV) have been enhanced for transport to inflammatory tissues. Blocking CXCR1 and CXCR2 in IGNVs significantly inhibited IGNV transport to inflammatory tissue. The therapeutic potential of IGNV was further demonstrated by enhancing the chemotherapeutic effect, as shown by the inhibition of tumor growth in two tumor models and the inhibition of the inflammatory effects of DSS-induced mouse colitis. The fact that IGNVs are capable of targeting inflammatory tissue and that there is an overexpression of chemokines in diseased human tissue provides a basis for the use of IGNV for the more targeted delivery of therapeutic agents to inflammatory sites and the use of IGNV as personalized medicine for the treatment of related inflammatory diseases.

Materials and procedures

Mice. C57BL / 6J mice, BALB / c mice and C.129S2 (B6) -Cxcr2tm1Mwm / J mice aged 6-8 weeks were obtained from Jackson Laboratories. All animal procedures were approved by the University of Louisville Institutional Animal Care and Utilization Committee.

Cell culture. Mouse T lymphoma EL4 cells, mouse 4T1, 4TO7 breast cancer cell lines, mouse NMuMG mammary gland epithelial cells, CT26 colon cancer and human umbilical vein endothelial cells (HUVEC) were purchased from ATCC. CT26 cells were grown in RPMI 1640 medium; the EL4, 4T1 and 4TO7 cells were maintained in DMEM medium supplemented with 10% heat inactivated FBS. HUVECs were cultured in complete endothelial cell growth medium (ECGM; Promocell # C-22010). NMuMG cells were cultured in complete DMEM medium supplemented with 10 pg / ml insulin. All cells were kept in a humidified CO2 incubator at 37 ° C.

Reagents and antibodies. Doxorubicin, curcumin, phorbol 12-myristate 13-acetate (PMA), lipopolysaccharide (LPS, Escherichia coli 0111: B4), PKH67, PKH26, and fluorescent cell linker kits were purchased from Sigma-Aldrich (St Louis, MO , U.S). Commercial DOX-NP® (300102S) and its control liposomes were purchased from Avanti Polar Lipids Inc. (Alabaster, Alabama, USA). Proteinase inhibitor cocktail tablets were obtained from Roche Diagnostics GmbH (Mannheim, Germany). DiR dye was obtained from Life Technologies (NY, USA). Sodium dextran sulfate (DSS, MW: 36,000-50,000) was obtained from MP Biomedicals, LLC (Santa Ana, CA). Infinitiy® ALT (GPT) and AST (GOT) stable liquid reagents were purchased from Thermo Scientific (Pittsburgh, PA, USA). The ATP luminescence detection system was obtained from PerkinElmer (Waltham, MA, USA). Recombinant mouse chemokines CXCL1, CXCL2, CXCL9, CXCL10, CXCL11, C ^c L2, and CCL5 were purchased from Biolegend (San Diego, CA, USA). Fluorescent conjugated antibodies against CCR3, CCR4, CCR5, CCR7, CCR9, CXCR2, CXCR3, mouse or human CXCR7, antibodies against CD3, F4 / 80, CD11b, CD11c, CD19, Ly6G and Mouse Gr1 were purchased from eBiosicence (San Diego, CA, United States). Antibodies against human CXCL1, CXCL10, CCL2, and CCL5 were obtained from LifeSpan BioSciences, Inc. (Seattle, WA, USA). Antibodies against mouse CXCL1, CXCL10, CCL2 and CCL5 and ELISA kits with CCL5, CXCL10 were purchased from Biolegend (San Diego, CA, USA). Secondary antibodies conjugated to Alexa Fluor 488 nm and 647 nm were obtained from Life Technologies (NY, USA). The human CCL2 ELISA kit was purchased from eBiosicence.

Isolation and purification of plasma membranes. Plasma membranes were isolated and purified using a procedure, as described above. Briefly, EL4 cells of the mouse lymphoma cell line with / without PMA stimulation (100ng / ml) were cultured for 12 hours. The cells (2 x 108) were then harvested and centrifuged at 500 x g for 10 minutes at 4 ° C. Cell pellets were resuspended in 1 ml of homogenization buffer at a final concentration of 10 mM Tris HCl, 25 mM D-sucrose, 1 mM MgCl2, 1 mM KCl, 10 pg / ml RNase, 10 pg / ml DNase, and 1 x proteinase inhibitor cocktail. The cell suspension was homogenized on ice by 100 passages using a handheld Dounce homogenizer. The supernatant was collected after centrifugation at 500 xg for 10 minutes. For further purification, the collected supernatants were subjected to a discontinuous sucrose density gradient centrifugation at 28,000 g for 45 minutes at 4 ° C in a 30%, 40% and 55% sucrose solution in a 0 saline solution. , 9%. For the isolation of the plasma membrane from mouse leukocytes or human peripheral blood, anticoagulant-treated peripheral blood was collected and centrifuged at 3,000 xg for 10 minutes at 4 ° C. Leukocytes were harvested and red blood cells were lysed by incubation with ACK lysis buffer (NH4Cl 8.024 g / L, KHCO31 g / L, EDTA-2Na 3.722 mg / L) for 5 minutes at 22 ° C. The procedure for lysis of red blood cells was repeated once. The leukocytes were then cultured in RPMI 1640 medium with LPS (100 ng / ml) for 12 hours prior to isolating and purifying the plasma membranes from mouse or human leukocytes with / without LPS stimulation using a gradient centrifugation procedure. discontinuous density of sucrose, as described above.

Preparation of plasma membrane derived vesicles. Plasma membranes purified from EL4 cells and mouse or human peripheral blood leukocytes were sonicated in a glass vial with 200 µl ddH2O for 10 minutes using an FS30D ultrasonic bath (Fisher Scientific). The resulting vesicles were subsequently extruded through 100 nm porous polycarbonate membranes using an Avanti mini-extruder (Avanti Polar Lipids).

Preparation of GNV coated with plasma membrane. Nanoparticles derived from grapefruit lipids (GNV) were prepared according to the protocol described above. To prepare plasma membrane coated VNG, 400 nmol of VNG was mixed with plasma membrane derived vesicles (approximately 5 x 105 cells) and extruded 20 times through a 200 nm porous polycarbonate membrane using an Avanti mini-extruder. NGVs coated with plasma membranes derived from EL4 or mouse or human leukocytes were hereinafter referred to as pseudo-inflammatory NGVs (IGNV).

Quantification of phosphorus. Phosphate was quantified in VNG using a standard phosphorus solution (0.65 mM, P3869-25 ml from Sigma). First, different amounts of phosphate standard solutions (50, 25, 10, 5 and 0 nmol) were prepared in 100 µl of ddH2O, then 30 µl of Mg (NO3) 2 were added and the mixture was heated to flame it until dry. The dried sample was dissolved in 300 µl of HCl and heated at 100 ° C for 15 minutes, cooled, and centrifuged at 1,000 rpm for 2 minutes. 700 µl of reaction buffer (1 part of 10% ascorbic acid and 6 parts of 0.42% ammonium molybdate in H2SO41 N) were added and the mixture was incubated at 45 ° C for 20 minutes. The absorption was read at OD280.

Critical micellar concentration (cmc) of GNV. Critical micellar concentration (CMC) is roughly defined as the lipid monomer concentration at which appreciable amounts of micellar aggregates begin first to appear in balance. Since VNG are not monomers and are composed of a set of lipids extracted from grapefruit nanoparticles, it was difficult to determine the CMC for each of the lipids derived from grapefruit nanoparticles that contribute to the formation of VNG. Therefore, unlike the usual approach used for the determination of the CMC, an alternative approach was taken to determine the total lipid concentration of VNG above which VNG is formed. Briefly, total lipids extracted from grapefruit nanoparticles were determined using the phosphate assay, as described above. Different volumes of total lipids (0-100 μl, 200 uM in stock solution) were pipetted into chloroform in glass tubes, and the solvent was removed under a stream of nitrogen, and the lipid films were subsequently kept under vacuum for 2 hours. The dried lipid films were hydrated at 60 ° C for 30 minutes in 20 mM HEPES buffer (pH 7.0). Following an ultrasonic bath (FS60 ultrasonic bath, Fisher Scientific, Pittsburg, PA) for 5 minutes, an equal volume of HEPES buffer (pH 7.0) was added and sonicated for another 5 minutes.

Analysis of the size distribution and Zeta potential. The size distribution and zeta potential of the particles were analyzed using a nano Zs Zetasizer (Malvern Instruments Ltd., Southborough, MA). For size measurement, particle samples were diluted in PBS and dispersed by sonication for 5 seconds prior to measurement. To determine the Zeta potential of the particles, VNG or VNG coated with a plasma membrane were washed in ddH2O by centrifugation at 100,000 xg for 45 minutes at 4 ° C. The samples were resuspended in 1 ml of ddH2O to measure the zeta potential of the particles.

FACS analysis of chemokine, integrin receptors on EL4 and IGNV cells. To test the expression of chemokine and integrin receptors, collected EL4 cells were washed and blocked with Fc blocker (2.4G2) at 4 ° C for 15 minutes and then stained with antibodies against CCR3, CCR4, CCR5, CCR7, Cc R9, CXCR2, CXCR3, CXCR7, a4-7 and LFA-1 labeled with PE at 4 ° C for 45 minutes. LFA-1 and chemokine receptors on IGNVs were quantitatively analyzed using a FACS procedure, as described above. Briefly, the GNV or IGNV were incubated with 4 mm diameter aldehyde / sulfate latex beads in 400 ml of PBS containing 2% FCS for 30 minutes, and then the mixtures were washed and subsequently stained. with PE-labeled antibodies against CCR3, CCR4, CCR5, CCR7, CCR9, CXCR2, CXCR3, CXCR7 and LFA-1 at 4 ° C for 45 minutes. Since a4p7 was not detected in EL4 cells; the levels of a4p7 in the IGNVs were not analyzed. All samples were analyzed using an Accuri C6 flow cytometer.

Chemokine detection in skin and tumor tissues. They were detected chemokines in normal tissue of the skin, inflammatory skin induced by LPS and tumor tissues CT26 and 4T1 using antibody arrays Proteome ProfilerTM (R & D System, Minneapolis, ^Mn, USA) according to the protocol of the manufacturer. Briefly, the tissues were excised and homogenized in PBS with 1x protease inhibitors and a final concentration of 1% Triton X-100. The samples were frozen at -80 ° C. After thawing, the supernatant of the samples was collected by centrifugation at 10,000 xg for 5 minutes at 4 ° C and the total protein was quantified using a NanoDrop 8000. After blocking for 1 hour, the membranes were incubated with a mixture of Panel A detection antibody cocktail of a reconstituted cytokine pool and the supernatant overnight at 4 ° C. After washing three times, the membranes were incubated with streptavidin-HRP for 30 minutes at 22 ° C. Then, after washing three more times, the membranes were incubated with 1 ml of Chemi Regent mix for 1-2 minutes at 22 ° C before being exposed to X-ray film for 1-5 minutes.

Obtaining images of the NGVs (IGNV) coated with plasma membrane. The purified IGNVs were prepared for electron microscopy using a standard procedure and were observed using an F20 FEI Tecnai electron microscope operating at 200 kV with 38,000 x magnification and 2.5 pm blur. Fresh prepared GNVs were also fixed and images were obtained as a control. Photomicrographs were taken with a Gatan Ultrascan 4000 CCD camera.

To further confirm the colocalization of the EL4 cell-derived plasma membrane with GNV, PKH67-labeled membrane-derived vesicles were mixed with PKH26-labeled GNV and then extruded 15-20 times through a porous polycarbonate membrane. 200 nm using an Avanti mini extruder. After washing at 100,000 xg for 30 minutes, the particles were resuspended and incubated with 4T1 cells for 12 hours in a 5% CO 2 incubator. The cells were then washed 3 times with PBS, fixed with 2% PFA for 10 minutes at 22 ° C, and permeabilized with 0.2% Triton X-100 for 2 minutes at 22 ° C. After washing 3 times, cells were stained with DAPI for 90 seconds. The colocalization of the plasma membrane derived from EL4 cells with GNV was examined using a confocal microscope equipped with a digital image analysis system (Pixera, San Diego, CA, United States).

Fluorescence resonance energy transfer analysis (FRET). The DiO / DPA system has been used for the detection of changes in the cytoplasmic membrane potential using the principle of fluorescence resonance energy transfer (FRET). This approach was adapted in the present studies by pairing the common tracer dye DiO (as a fluorescent donor) with dipichrilamine (DPA), an anion low molecular weight lipophilic, as a non-fluorescent acceptor. As a donor, DiO is a bright, non-toxic membrane marker that allows repeated imaging of fluorescently labeled VNG. However, if DiO is close to the DPA-dye-labeled activated T-cell plasma membrane, which allows fluorescence arrest, the result is a detected non-fluorescent signal. To test whether IGNVs are coated with activated T-cell plasma membrane, DiO-labeled GNV (5 mM) and DPA-labeled EL4 cell plasma membrane-derived vesicles (5 mM) were prepared by sonication using a procedure, as described described above. The extrusion of VNG with EL4-derived membrane vesicles was carried out by extrusion 20 times through a porous 20 nm polycarbonate membrane using an Avanti mini-extruder. The mixture of DiO-labeled GNV (5 mM) and DPA-labeled EL4 cell membrane-derived vesicles (5 mM) without extrusion was used as a control. Both samples were then diluted (1: 5, 1:10, 1:20, 1:50, 1: 100 and 1: 150) with ddH2O and the fluorescence intensity of the DiO dye was measured by a plate reader. fluorescent (Biotek HTS multi-mode readers). The non-quenched fluorescent signal, expressed as% fluorescence intensity, was determined as follows: [(Fluorescence intensity of the diluted sample - Fluorescence intensity of the undiluted sample) / Fluorescence intensity of the diluted sample ] x 100%. Results represent the mean of three independent experiments (error bars, standard errors of the means).

Transwell Assay. HUVEC cells were seeded in Costar transwell inserts ("Transwell") (6.5 mm diameter and 5 mm pore size, Corning, Corning Incorporated, NY, USA) coated with fibronectin (7.5 mg / cm2) at a cell density of 5x104 cells / well. Chemokines were placed in the lower chamber. NGVs labeled with PKH26, IGNV, chemokines, or IGNV preincubated with LFA-1 antibody were added to the upper chamber and incubated with HUVEC cells at 37 ° C for 24 and 48 hours. After incubation, the media in the lower chamber was collected and diluted. The fluorescence intensity of PKH26-labeled g Nv or IGNV was measured by a fluorescent plate reader (Biotek HTS multi-mode readers). The fluorescence intensity of GNV or IGNV labeled with PKH26, expressed as the% efficiency in transwell of the fluorescence intensity of GNV or IGNV labeled with PKH26, was determined as follows: (fluorescence intensity of PKH26 of the lower well in the time samples were collected / total PKH26 fluorescence intensity of GNV or PKH26-labeled IGNV added to the top well of the chamber at the beginning of the culture) x 100%. The media in the lower chamber was collected and centrifuged. The particles were then resuspended in ddH2O, spread on a slide, and stained with 4 ', 6-diamidino-2-phenylindole (DAPI). The number of particles associated with HUVEC cells in the lower chamber was estimated by counting 10 randomly selected fields (x20 magnification) using ImageJ software. None of the samples examined was positive for DAPI staining.

Hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC). Livers, lungs, spleens, kidneys of BALB / c mice treated with PBS or IGNV (200 nmol and 800 nmol, IV injection three times) and colon tissues of mice fed DSS were fixed overnight in 4% paraformaldehyde and embedded in paraffin; then 5 µm sections of tissues were stained with H&E.

Human breast cancer tissues and colon cancer tissues were collected at the First People Hospital in Huai'an China. All patients provided their written consent. The use of human tissues in this study was approved by the institutional review committee of the Huai'an First People Hospital of China and was carried out in accordance with the international guidelines for the use of human tissues. Paraffin-embedded tissue sections (5 pm) from breast cancer, colon cancer and adjacent were rehydrated and heated in antigen retrieval solution for 45 minutes. Endogenous peroxidase activity was inhibited by incubation with 3% hydrogen peroxide for 10 minutes at 22 ° C and non-specific sites were blocked with 5% BSA for 45 minutes. Slides sectioned tissues were then incubated with primary antibodies [(polyclonal antibody against CXCL1 (0.15 pg / ml), polyclonal antibody against CXCL10 (1 pg / ml), antibody against CCL2 (1 pg / ml) and CCL5 (5 pg / ml)] overnight at 4 ° C. Sections were processed with an appropriate biotinylated secondary antibody and a streptavidin biotin peroxidase amplification kit (Vectastain, Vector Laboratories, Burlingame, cA). The reaction of the Peroxidase was finally developed with diaminobenzidine (Dako) and sections counterstained with Mayer's hematoxylin. Slides counterstained with weak Mayer's hematoxylin solution for 2 minutes. Negative control slides were prepared without addition of primary antibody. Grade Chemokine expression was independently recorded by two expert observers as the percentage of cells positive for chemokine based on a visual assessment of the intensity of pr Brown reaction product within the cytoplasmic regions of each image on a scale of 0 (no staining) to 3+ (strong staining). To quantify the intensity of the staining, an average score was then calculated by summing the individual intensity level scores.

In vivo image. To assess the stability of circulating IGNV in mice, 200 nmol DiR-dye-labeled IGNV was injected into mice through the tail vein. Blood was drawn into an anticoagulant tube at various time points (3 hours, 24 hours, 48 hours, 72 hours, and 120 hours) after injection. The intensity of the DiR signals from blood samples of equal volume was then measured using a Kodak Image Station (4000MM Pro system, Carestream, Woodbridge, CT) and quantified using Carestream MI software.

To monitor IGNVs in LPS-induced acute skin inflammation of mice, I.V. DiR dye-labeled IGNVs (20, 40, 150, 300 nmol) in mice. At 6 hours and 24 hours after injection, live mice were imaged using a Kodak Image Station. The inflamed skin was then removed 24 hours after IGNV administration and DiR dye signals in the skin were quantified.

To study the targeted delivery of IGNV in mice with DSS-induced colitis, they were injected I.V. IGNV or GNV labeled with DiR dye (200 nmol) in normal mice or mice with colitis that had been given 2.5% DSS in their drinking water for 5 days. 24 hours after a single IV injection, colons were harvested and scanned using a Kodak Image Station and quantified using vendor software.

For the biodistribution of IGNV in tumor-bearing mice, mice bearing mouse colon tumor (CT26) and mouse breast tumor (4T1) were administered I.V. with IGNV (200 nmol) or GNV (200 nmol) labeled with DiR dye. Whole body imaging was performed at 6 hours and 24 hours after injection. DiR dye signals in CT26 and 4T1 tumor tissues were also quantitatively measured 24 hours after injection.

To determine the effects of chemokines on IGNV transport, 200 nmol of IGNV labeled with DiR dye, or 100 ng of each of the recombinant chemokines, that is, CXCL1, CXCL2, CXCL9, CXCL10, were preincubated with tissue extract. CCL2, CCL5 or combinations of CXCL1 / 2, CXCL9 / 10, CCL2 / 5 or CXCL1 / 2/9/10 plus CCL2 / 5 overnight at 4 ° C. The IGNVs were then washed at 100,000 g for 20 minutes at 4 ° C, resuspended in 100 p.l PBS and injected into mice with acute LPS-induced skin inflammation or CT26 tumor-bearing mice. 24 hours after injection, skin or tumors were removed and DiR dye signals were quantitatively measured using a Kodak Image Station and quantified using vendor software.

Cytokine assays. To assess the potential toxicity of IGNV and commercial liposomes, I.V. 200 nmol and 800 nmol of IGNV or commercial liposomes as controls in BALB / c mice daily for 3 days.

24 hours after the last injection, peripheral blood was collected and cytokines (TNF-α, IL-6 and IL-1P) were measured using ELISA kits.

To determine the effect of curcumin supplied by IGNV in inhibiting the induction of TNF-a, IL-6 and IL-1p, colon tissues of mice with DSS-induced colitis were cultured ex vivo and TNF-a were quantified, IL-6 and IL-1 p. Colon tissues of mice with DSS-induced colitis previously injected with I.V. PBS, GNV (200 nmol), IGNV (200 nmol), curcumin (50 mg / kg), GNV-Cur (50 mg / kg) or IGNV-Cur (50 mg / kg) every 2 days 5 times were collected for the ex vivo culture. Briefly, the most distal 2 cm of the colon was washed with PBS containing penicillin / streptomycin, and then further cut into 1 cm 2 sections. Colon sections were cultured in serum-free RPMI 1640 medium supplemented with penicillin / streptomycin. After 24 hours of culture, cell-free supernatants were collected and cytokine secretion was analyzed using ELISA kits (eBioscience).

To measure human chemokines released in SW620 human colon cancer cell culture supernatants, 2 x 105 cells / well were grown in 6-well plates. After 48 hours in culture, the medium was harvested and CCL2, CCL5 and CXCL10 were analyzed using Biolegend's "deluxe" ELISA MAX® kit.

Induction of local inflammation of the skin. Dermal inflammation of C57BL / 6j mice (body weight = 25 to 30 g) was induced by a single intradermal injection in the flank region of 30 pg of LPS (Escherichia coli 0111: B4) in 50 µl of isotonic saline. 6 hours after injection, mice with a visible local skin inflammatory response were recruited for this study.

DSS-induced colitis model. Colitis was induced using 2.5% (w / v) sodium dextran sulfate that had been added to the drinking water. The DSS solution was prepared fresh every other day. To evaluate the therapeutic effects of curcumin delivered by Ig Nv in colitis, beginning on day 3 after the mice were provided with 2.5% DSS in their drinking water, the mice were injected I.V. with PBS, free GNV (200 nmol), IGNV (200 nmol), free curcumin (50 mg / kg), curcumin-loaded GNV (50 mg / kg) or curcumin-loaded IGNV (50 mg / kg) every 2 days for a total of 5 injections. Body weight and the presence of blood in the stool were monitored daily.

Measurement of curcumin concentration in mouse colon tissues. Mice with DSS-induced colitis were injected IV with PBS, GNV, IGNV, free curcumin, GNV-Cur or IGNV-Cur every 2 days, curcumin in colon tissues was quantitatively analyzed using high performance liquid chromatography (HPLC) , as previously described. Briefly, colon tissues were harvested 6 hours after the last injection, weighed and placed in 1 ml of PBS and homogenized. Fifty µl of solution was added citrate buffer to 0.8 ml aliquots of homogenized samples and vortexed for 30 seconds, followed by the addition of 2 ml of ethyl acetate. The supernatants were then collected after centrifugation at 1,000 xg for 5 minutes and dried under a stream of nitrogen gas. The solid samples obtained were redissolved in 100 µl of methanol and centrifuged at 10,000 xg for 10 minutes. Twenty µl of supernatant was used for HPLC analysis. Curcumin was detected by reverse phase HPLC with a DAD detector (Agilent 1100, USA) at a flow rate of 1 ml / min. The solvent system was (A) ddH2O and (B) acetonitrile containing 0.1% TFA. The following mobile phase B gradients were used to develop the column: 10-50% for 0-18 minutes, 50-90% for 18-25 minutes, 90% for 25-30 minutes, and 90-10% for 30-35 minutes.

Mouse models with CT26 and 4T1 tumors. Xenograft tumor growth models were used to demonstrate IGNV-mediated targeted delivery of chemotherapy drugs to tumors versus free doxorubicin (free DOX) or GNVDOX. In the first series of experiments, six week old female BALB / c mice were injected subcutaneously with the murine colon cancer cell line CT26 (5.0 x 10 5 cells / mouse in 50 µl PBS). In a second set of experiments, six week old female BALB / c mice were injected into a mammary fat pad with the murine breast tumor cell line 4T1 (5.0 x 105 cells / mouse in 50 μl PBS) . When the tumors reached approximately 60 mm3 in volume, the mice were randomly assigned to different treatment groups and injected I.V. with free GNV, IGNV, doxorubicin (DOX, 100 pg), DOX loaded GNV (GNV-DOX, 100 pg DOX) or DOX loaded IGNV (IGNV-DOX, 100 pg DOX). Mice were treated every 3 days for 30 days. The growth of the tumors was measured using a procedure, as described above.

Charging efficiency. To evaluate the efficiency of loading doxorubicin and curcumin in IGNV, GNV-DOX or GNV-Cur particles were prepared by adding 200 pg of doxorubicin or curcumin in a grapefruit lipid film and subsequently sonicated using a procedure , as described above. To further manufacture IGNV-DOX or IGNV-Cur, the particles of GNV-DOX or GNV-Cur formed by ultrasound, as described above, were mixed with plasma membrane-derived vesicles of EL4 cells and then extruded 15-20 times through an Avanti mini extruder with a 200nm porous polycarbonate membrane. The resulting suspension was centrifuged at 100,000 x g for 30 minutes. The supernatants were collected and the residual doxorubicin or curcumin content was measured using a CARY 100 Bio UV-Visible spectrophotometer at a wavelength of 497 and 426 nm, respectively. The loading efficiency was calculated as follows: Loading efficiency = (total drug - free drug) / total drug x 100%.

Chemotherapy drug release profile. To test the in vitro profile of doxorubicin released from IGNV-DOX or DOX-NP® purchased from Avanti Polar Lipids Inc. (Alabaster, Alabama, United States), IGNV-DOX or DOX-NP® were suspended with 200 pg of doxorubicin in 1 ml of PBS buffer solution at different pH values of 5.0, 5.5, 6.0, 6.5 and 7.2. Each suspension was placed at 37 ° C with shaking. At a predetermined time (0.5, 1, 2, 3, 6 and 24 hours), the suspensions were centrifuged at 100,000 x g for 30 minutes. The amount of doxorubicin released from IGNVDOX or DOX-NP® was measured spectrophotometrically at 497 nm. Curcumin release from IGNV-Cur was also analyzed using a set of UV-Visible spectrophotometers at 426 nm.

ATPlite assay. To test the potential cytotoxicity of IGNVs and commercial DOX-NP® control liposomes, 4T1 cells were grown in 24-well culture plates for 24 hours at different doses (0, 10, 20, 40, 80, 160 nmol). of IGNV or control liposomes from Avanti Polar Lipids Inc. (Alabaster, Alabama, USA). The cellular cytotoxicity of the IGNVs was measured using an ATP luminescence detection assay system (PerkinElmer, MA, USA). Briefly, 200 µl of mammalian cell lysis buffer was added to each well and the plate was shaken for 5 minutes. 50 µl of cell lysis was dispensed onto an OptiPlate, mixed with 50 µl of substrate solution, the plate was shaken at 700 rpm for 5 minutes, and luminescence was measured using a BioTek Synergy® HT multi-detection microplate reader with software. Gen 5 data analysis version 1.08 (Winooski, VT, United States).

Statistic analysis. A one-tailed analysis of variance (ANOVA) followed by Turkey Post Hoc tests was used to determine the differences that occurred between groups and the T test was used to determine the difference between two groups (* p <0.05, ** p <0.01 and *** p <0.001).

Example 1 - Characterization of GNV coated with activated T cell membrane fraction enriched in inflammatory chemokine receptor (IGNV).

IGNV was generated by the binding of membranes derived from PMA activated EL4 T cells to GNV. The morphology of IGNV (Figure 6A) and the amount of chemokine receptors (Figure 6B) of EL4 T cells stimulated with / without PMA in the IGNVs were analyzed. Purified middle band vesicles from a sucrose gradient (Figure 7) were prepared by extrusion and subsequently joined with GNV. Next, the Zeta potential and the size distribution of IGNV were analyzed (Figure 1B). Transmission electron microscopy (TEM) imaging (Figure 1C) indicated that IGNVs shared a similar morphology with GNV. The IGNV were internalized by CT26 colon cancer cells in a similar way to GNV (Figure 1D) and co-localized with the EL4 membrane (Figure 1D, last column). Fluorescence resonance energy transfer (FRET) analysis further confirmed that more than 83 ± 2.2% of the GNVs were coated with the plasma membrane of activated EL4 T cells (IGNV) (Figure 1E). After preparation and for five days at 22 ° C (Figure 9A) or 25 hours at 37 ° C (Figure 9B), the IGNVs were stable without significant changes in size or loading.

Example 2 - Directed migration of IGNV to inflammatory sites.

Next, to determine whether the IGNVs would leave the peripheral circulation and target inflammatory tissues, three inflammatory mouse models were tested. We first searched to determine whether IGNV transmigrated through a HUVEC monolayer with greater efficiency than GNV using an in vitro transwell assay. The data demonstrated that a greater number of IGNV-transfected HUVECs (DAPI + PKH26 +) migrated to the bottom of the transwell (right panel) than VNGs (left panel) over a period of 48 hours (Figure 2A). The addition of chemokines (CXCL1 / 2/9/10, CCL2 / 5) to the bottom of the transwell further improved the transmigration efficiency of IGNV (Figure 2A, right panel, 4th column, ** p <0.01). The improved directed migration of IGNV to inflammatory sites compared to GNV was further confirmed in different inflammatory models, two models of acute inflammation (an LPS-induced skin inflammation (Figure 2B) and DSS-induced colitis (Figure 2C, * * p <0.01)) and two chronic inflammatory cancer models (CT26 colon cancer (Figure 2D, *** p <0.001) and 4T1 breast cancer (Figure 2E, *** p <0.001)).

Example 3 - Chemokine and chemokine receptors play a role in the targeted migration of IGNV.

Recruitment of leukocytes into inflamed tissue follows a well-defined cascade of events, beginning with the capture of free-flowing leukocytes to the vessel wall, followed by coiling, adhesion to endothelial cells, strengthening after adhesion, entrainment, and finally , transmigration through endothelial junctions at sites of inflammation. During these stages, chemokines / chemokine receptors play a key role in the last stage, transmigration. Therefore, the chemokine profiles of the inflammatory tissue extracts and the types of chemokine receptors that coat the IGNVs were analyzed. The chemokine cluster data (Figure 3A) indicated that the identified chemokines are at much higher concentrations in the extracts from the inflammation models that were tested than the non-tumor mammary gland extracts (Figure 3A, normal). It was also observed, in general, that stronger chemokine signals were detected in 4T1 breast tumor extracts than in LPS-induced skin inflammation or CT26 colon tumor skin. Data from the FACS analysis indicated that the corresponding chemokine receptors were detected in the IGNVs (Figure 3B). To determine which chemokine or chemokines in inflammatory tissues play a causal role in the recruitment of IGNV into inflammatory tissue, an in vitro transwell assay was performed. The results indicated that IGNV transmigration was markedly affected by the addition of chemokines at the bottom of the transwells; while there was no change in the migration of GNV with the addition of chemokines. The addition of CXCL1 or CXCL2 resulted in more IGNV detected in the bottom of the well 48 hours after the addition and the combination of chemokines, CXCL1 / 2/9/10, CCL2 / 5, led to the highest efficiency in transmigration of IGNV (figure 10). The effects of chemokines on I ^g N ^v transmigration were then confirmed by results generated from a chemokine neutralization assay. Although preincubation with recombinant chemokine against each chemokine receptor partially attenuated IGNV migration, neutralization of the six indicated chemokine receptors or preincubation with 4T1 tumor extract led to a maximal reduction in IGNV transmigration (Figure 3C , * p <0.05 ** p <0.01 and *** p <0.001). This reduction was also confirmed in a mouse model of LPS-induced skin inflammation, in which mice were injected IV with DiR dye-labeled IGNV that had been pre-incubated with 4T1 tumor extract (Figure 3D, ** p < 0.01).

Data generated from a 24-hour in vivo image analysis (evolution over time was determined based on the data in Figure 3D) further confirmed that although CXCL1, CCL2 and CCL5 have an effect, CXCL2 plays a role. key in the targeted migration of IGNV to inflammatory sites, including acute inflammation in the skin induced by LPS (Figure 3F, * p <0.05, ** p <0.01 and *** p <0.001) and the model colon cancer CT26 (Figure 3G, * p <0.05, ** p <0.01 and * "p <0.001).

Integrins, including LFA-1 and a4p7, have multiple roles in the leukocyte recruitment process from initial adhesion to vessel walls to entrainment before the final stage, transmigration. The FACS data indicated that although a4p7 was not detected, LFA-1 was highly expressed in PMA-activated EL4 cells (Figure 11) and was present in Ig Nv (Figure 12). Transmigration of IGNVs was dramatically reduced in vitro (Figure 3H, *** p <0.001) and in vivo (Figure 3I, * p <0.05) when LFA-1 was neutralized in IGNVs. Since multiple factors, including LFA-1, play a role in the process of directed migration of leukocytes and nanoparticles to inflammatory sites, and chemokines / chemokine receptors are known to play important roles in the last stage (transmigration) of transport of leukocytes to inflammatory tissue, in general, chemokine-related assays were used as a major determinant of function without further analysis of the role of LFA-1 in IGNV.

It was further determined whether the chemokines of interest were up-regulated in human cancer tissues. The results of immunohistochemical chemokine staining in human colon cancer (Table 1) and breast cancer (Table 2) suggested a much higher expression of CXCL1, CXCL10, CCL2, and CCL5 in tumor tissues than in adjacent non-tumor tissues (Figure 3E, tables 3, 4).

Table 1. Characteristics of 20 patients with colon cancer.

Sex

male 13

female 7

Age

male 59.85 ± 3.027

female 56.88 ± 4.129

Differentiation

good / moderate 14

bad 6

Lymph node involvement

yes 14

No 6

Table 2. Characteristics of 21 patients with breast cancer.

\1 Features

\

Cases

Age 54.81 ± 2.126

Lymph node involvement

yes 9

no 12

Table 3. IHC scores of chemokines (CXCL1, CXCL10, CCL2 and CCL5) in human colon cancer tissues.

Niveles de expresión

Chemokines

Levels of expression

CXCL1 + ++

Adjacent 8

Tumor tissue 2 6 12

CXCL10

Tumor tissue 12 8

Table 4. IHC Ratings of Chemokines (CXCL1, CXCL10, CCL2, and CCL5) in Human Breast Cancer Tissues

Niveles de expresión

Chemokines

Levels of expression

CXCL1 + ++

Adjacent 8

Tumor tissue 3 6 12

CXCL10

Adjacent 18 3

Tumor tissue 2 10 9

CCL2

Adjacent 13 8

Tumor tissue 5 16

CCL5

Adjacent 18 3

Tumor tissue 8 12

To further demonstrate whether the approach described above could be applied for the treatment of patients in a personalized manner, membrane-coated VNG was purified from LPS-stimulated leukocytes isolated from the peripheral blood of healthy human individuals (Figure 13A) or from mice ( Figure 13B) using a sucrose gradient. The chemokine receptors on the IGNVs were quantitatively analyzed (Figures 4A-4B). Next, it was determined whether IGNVs were capable of targeting a human tumor using the human colon tumor model SW620, since the chemokines of interest are overexpressed in human colon cancer, as well as in tissue from breast tumor, and are also released from SW620 colon cancer cells (Figure 14). The results of in vivo imaging analyzes indicated that human SW620 tumor bearing mice (Figure 4C, ** p <0.01) or mice locally challenged with LPS (Figure 4D, * p <0.05 and * ** p <0.001) attracted more IGNV than GNV, and IGNV coated with purified membranes of LPS-stimulated leukocytes have the highest fluorescence intensity on day 5 after IV injection The results of an in vitro transwell assay (Figure 3C) elicited a further determination of whether CXCR2 plays a dominant role in the targeted migration of IGNV to the inflammatory site. Results generated from in vivo imaging analysis indicated that the membrane-coated IGNV of LPS-stimulated leukocytes isolated from the peripheral blood of CXCR2 knockout mice had significantly attenuated IGNV migration to the inflammatory site (Figure 4E ), suggesting that CXCR2 in IGNV plays a key role in the targeted migration of IGNV.

Example 4 - In vivo therapeutic effects of drugs carried by IGNV.

Since no adverse side effects have been observed with an I.V. of IGNV (Figures 15A-15F), it was tested whether IGNV can be used as a therapeutic drug delivery vehicle. IGNVs are capable of being loaded with different drugs, including chemotherapy drugs, such as doxorubicin, and anti-inflammatory agents, such as curcumin (Figure 16A). Both doxorubicin and curcumin loaded IGNVs have similar Zeta potential and size distribution (Figure 16B, 16C), although the loading capacity (Figure 16D) and release of an agent (Figure 16E) is different for different types of agents. . Further analysis of the stability of the IGNVs indicated that the circulating IGNVs were stable and detectable until day 5 (Figure 5A) after an IV injection, which in turn provides a longer period of time for the IGNV to migrate. directed to where the inflammation is occurring. Furthermore, IGNV-DOX were stable without releasing doxorubicin until they were at a pH of 5.5 (Figure 5B, ** p <0.01) which is the pH in tumor tissue. In contrast, a pH as low as 5.0 did not result in the release of doxorubicin from commercially available liposomes loaded with doxorubicin (Figure 17A), although no differences were observed in cellular cytotoxicity (Figure 17B), induction in vivo of proinflammatory cytokines (Figure 17C) and liver enzyme release (Figure 17D) between IGNV (Figure 14) and control liposomes. The tissue distribution of IGNV-encapsulated doxorubicin was then determined. The doxorubicin concentration was higher in the tumor and lower in the liver of tumor-bearing mice injected I.V. with IGNV-DOX when compared to mice treated with DOX-NP® (Figure 5C, * p <0.05). This result was further confirmed by the much stronger intensity of doxorubicin signals detected in CT26 tumor bearing mice as well as 4T1 breast tumor bearing mice injected IV with IGNV-DOX compared to mice treated with GNV-DOX or free doxorubicin (Figure 5D). Injection of GNV-DOX mixed with EL4 cell-derived membrane vesicles presented significantly lower levels of doxorubicin detected in the 4T1 tumor than in IGNV-DOX (Figure 18), suggesting that the extrusion of GNV with the leukocyte membranes activated, leading to the formation of IGNV, is necessary for more efficient delivery of DOX to inflammatory sites. The biological effects of IGNV-DOX in the CT26 colon and 4T1 breast tumor models were also significant compared to the other treatments. Treatment with IGNV-DOX led to a significant inhibition in the growth of the CT26 tumor and the 4T1 tumor (Figure 5E, * p <0.05, ** p <0.01 and *** p <0.001) and extended the survival of tumor-bearing mice (Figure 5F, * p <0.05, ** p <0.01). The results of the IGNV-Cur treatment of mice with DSS-induced colitis indicated that IGNVs containing curcumin had a better therapeutic effect on inhibiting colitis than GNVs containing curcumin or curcumin alone. This conclusion is supported by the fact that there was less blood in the stool (Figure 19A), fewer leukocytes were seen infiltrating HE-stained mouse colon tissue (Figure 19B), there was less weight loss (Figure 19C), and there was a significantly improved survival rate (figure 19D) in mice with dSS-induced colitis injected IV with IGNV-Cur than the indicated control groups. These results were also consistent with a higher concentration of curcumin detected in the colon tissues of mice treated with IGNV-Cur (Figure 19E). ELISA analysis further indicated that significantly less TNF-α, IL-6 and IL-1P were detected in extracts of colon tissue from mice with DSS-induced colitis injected I.V. with IGNV-Cur than with PBS / DSS, free Cur or GNV-Cur (figure 20).

Discussion of Examples 1-4.

The above study describes an approach for the targeted delivery of therapeutic agents to inflammatory sites where appropriate cells are recognized; thereby promoting many more substantial therapeutic benefits without inducing adverse side effects. It was shown that IGNVs can be an effective, personalized approach to potentially treating patients with a variety of inflammatory conditions. The use of IGNV avoids several of the problems that have arisen with conventional therapy vectors, such as lack of specificity in tissue recognition, immunogenicity, difficulty in scalability and production, need for lifelong follow-up of tumorigenesis and other adverse clinical outcomes. Since IGNVs do not cause these problems, they have great potential as targeted delivery vehicles, in particular as VNG production is easily scaled up and NGVs can be coated with leukocyte membranes from an individual, making this approach personalized and economically viable for the treatment of patients in low- and middle-income countries that do not have complete facilities to manufacture synthetic therapeutic vectors.

To successfully recognize a specific tissue, at least four objectives must be met: 1) extended circulation of the delivery vector, 2) penetration of the tissue by the vector, 3) tissue specificity, and 4) release of payload, ie that is, the therapeutic agent. Tumor tissues have abnormal fluid and molecular transport dynamics, especially for macromolecular drugs. This phenomenon is known as "enhanced permeability and retention effect (EPR)" of macromolecules and lipids in solid tumors. Therefore, the longer the vector is in the circulation, the greater the opportunity it has to penetrate tumor tissue by utilizing the EPR effect. Previously published work shows that a nanovector composed of grapefruit nanoparticle lipids circulates in peripheral blood for more than 5 days in tumor-bearing mice. GNVs are stable in the circulatory system and thus have a greater opportunity to enter tumor tissue. Furthermore, the critical concentration of lipids derived from grapefruit nanoparticles for the formation of GNV was 1.5 | jMI (figure 21) and the GNV did not increase in size with increasing concentrations of total lipids of up to 5 mM. The diameter of the GNVs at a concentration of 25 mM-50 mM and at 25 ° C increases to 200 nm, and remains at 200 nm, while the lipid concentration increased further up to 100 mM. It was believed that this finding not only provides a guide for the selection of concentrations of lipid derived from grapefruit nanoparticles to make VNG of the desired size (50-200 nm), but also a wide range of concentrations of lipid derived from nanoparticles can be selected. of grapefruit for the manufacture of CNG without leading to an additional increase in the size of CNG. This last characteristic has an advantage in case a large amount of lipids derived from grapefruit nanoparticles are needed to manufacture the GNV, which allows higher concentrations of therapeutic agents to be encapsulated by the GNV. However, this characteristic of GNVs alone may not be sufficient to deliver a broad agent to the target tissue to have a therapeutic effect. Getting a specific and safe delivery of a drug through endothelial cells is essential if it is to reach the target tissue. However, due to the fact that most delivery vectors lack an affinity for the endothelium, only a small fraction of the therapeutic agents can be delivered to the target tissue using the EPR effect. In the previous study, the IGNVs were modified so that they could take advantage of the activated leukocyte pathway that is driven primarily by chemokines / chemokine receptors by coating the IGNVs with activated leukocyte membranes. This modification of the IGNVs significantly improved their transmigration capacity in endothelial cells, so that the IGNVs could enter inflammatory tissues. As demonstrated in this study, this strategy can not only be applied to breast cancer and colon cancer, but there is the possibility that IGNVs can be used to treat many different types of diseases, since the inflammatory process is a feature of many chronic diseases, including cancer, infectious diseases, and autoimmune diseases. As shown in this study, the chemotherapy drug doxorubicin and the anti-inflammatory agent curcumin can be successfully delivered by the IGNVs to reach the desired inflammatory site and achieve a therapeutic effect by modifying the GNVs with activated leukocyte membranes from individuals. . Specifically, it was shown that the I.V. IGNV-DOX or IGNV-Cur significantly improves growth inhibition of breast tumor and colon tumor, and attenuates DSS-induced colitis, respectively. This response was due to the improved recruitment of IGNV into the tumor, as well as the inflamed colon tissue. Activated leukocyte membranes were required to gain this advantage, as it allows the delivery of IGNV at the precise location where inflammation is occurring.

It was also thought that factors other than chemokines / chemokine receptors may play a role in the transport of IGNVs to an inflammatory site. Previous studies have suggested that LFA-1 plays a role in transporting nanoparticles to an inflamed site. The present studies used LFA-1 (CD11a-CD18) as an example and the above data also showed that CXCR2 and LFA-1 played a role in the transmigration of IGNVs, as demonstrated in an in vitro transwell blocking assay and an inflammatory mouse model of the skin in vivo. Therefore, the role of the IGNV chemokine receptors, as demonstrated in this study, does not exclude a set of other IGNV factors, such as CXCR2 and LFA-1, which also play a role in the recruitment process of IGNV in inflamed tissue. This is also a reason why it was believed that IGNV coated with total leukocyte membranes may have a greater potential for application in personalized medicine for targeted delivery to inflammatory sites than the use of IGNV coated with chemokine receptors. individual. It was conceivable, furthermore, that the membrane-associated chemokine and integrin profiles of circulating inflammatory cells from patients with different chronic inflammatory diseases may be different in composition. Additionally, individual chemokine receptor coated GNVs can be potentially difficult to optimize in combinations or as a customized set or group of chemokine receptors that are best suited for targeted delivery to an individual patient. In addition, there may be a higher cost for the production of recombinant chemokine receptors and recombinant production would require FDA approval for the clinical use. Finally, potential biosafety problems could arise due to the use of synthesized recombinant chemokine receptors.

A suitable delivery vehicle must be susceptible to manipulation so that the delivered therapeutic drug can be released into the target tissue. Evidence accumulated in recent decades has shown that the pH in an electrode-evaluated human tumor is, on average, lower than the pH of normal tissues. The results presented in the previous study show that doxorubicin encapsulated in IGNVs is stable until the pH falls to 6.0 or below. This characteristic of IGNVs allows the encapsulated drug to be selectively released into tumor tissue and therefore reduces the side effects seen when chemotherapy treatment affects healthy organs and tissues in a non-discriminatory way, which is one of the Major obstacles to chemotherapy for treating cancer patients.

In recent decades, substantial experimental and clinical evidence supports the conclusion that one of the most important mechanisms operating in tumor progression involves chemokines and their receptors. Chemokines and their receptors not only play a role in cancer-related inflammation, but have also been implicated in the invasion and metastasis of various types of cancer. Without wishing to be bound by any particular theory, it is believed that pseudo-inflammatory chemokine receptors delivered by GNV may also act as soluble receptors to block the chemokine receptor-mediated pathway (s) expressed on tumor cells or other tumor-associated cells.

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It will be understood that various details of the subject matter disclosed herein may be changed without departing from the scope of the subject matter disclosed herein. Furthermore, the above description is for the purpose of illustration only and not for the purpose of limitation.

Claims (14)

1. Composition, comprising: a microvesicle derived from an edible plant; and a plasma membrane that lines the microvesicle, the plasma membrane derived from a recognition cell. Composition according to claim 1, in which the microvesicle encapsulates a therapeutic agent and / or in which the recognition cell is an activated leukocyte, and / or in which the edible plant is a fruit or a vegetable, in which, optionally, the fruit is selected from a grape, a grapefruit and a tomato. Composition according to claim 2, in which the therapeutic agent is selected from a phytochemical agent and a chemotherapeutic agent, in which, when the therapeutic agent is a phytochemical agent, the phytochemical agent is optionally selected from curcumin, resveratrol, baicalein, equol, fisetin and quercetin, and wherein, when the therapeutic agent is a chemotherapeutic agent, the chemotherapeutic agent is optionally selected from the group comprising retinoic acid, 5-fluorouracil, vincristine, actinomycin D, adriamycin, cisplatin, docetaxel, doxorubicin, and taxol. Composition according to claim 1, in which the therapeutic agent comprises a nucleic acid molecule selected from a siRNA, a microRNA and a mammalian expression vector. 5. A pharmaceutical composition, comprising a composition according to claim 1, and a pharmaceutically acceptable vehicle, carrier or excipient. Composition according to claim 1 or claim 2, for use in the treatment of an inflammatory disorder. A composition for use according to claim 6, wherein the inflammatory disorder is selected from the group comprising sepsis, septic shock, colitis, colon cancer and arthritis. 8. A composition for use according to claim 6, wherein the composition is administered orally or intranasally. A composition for use according to claim 6, wherein the administration of the composition reduces the amount of an inflammatory cytokine in an individual, wherein optionally the inflammatory cytokine is selected from the group comprising factor of Tumor necrosis-a, interleukin-1 p, interferon and interleukin-6. A composition for use according to claim 6, in which the therapeutic agent is a phytochemical agent, such as curcumin. Composition for use according to claim 6, in which the inflammatory disorder is colitis. Composition according to claim 1 or claim 2, for use in treating cancer in an individual. Composition for use according to claim 12, in which the therapeutic agent is selected from a phytochemical agent and a chemotherapeutic agent. Composition for use according to claim 12, in which the cancer is selected from a brain cancer, a breast cancer, a lung cancer and a colon cancer.